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1.  DRD2/CHRNA5 Interaction on Prefrontal Biology and Physiology during Working Memory 
PLoS ONE  2014;9(5):e95997.
Background
Prefrontal behavior and activity in humans are heritable. Studies in animals demonstrate an interaction between dopamine D2 receptors and nicotinic acetylcholine receptors on prefrontal behavior but evidence in humans is weak. Therefore, we hypothesize that genetic variation regulating dopamine D2 and nicotinic acetylcholine receptor signaling impact prefrontal cortex activity and related cognition. To test this hypothesis in humans, we explored the interaction between functional genetic variants in the D2 receptor gene (DRD2, rs1076560) and in the nicotinic receptor α5 gene (CHRNA5, rs16969968) on both dorsolateral prefrontal cortex mediated behavior and physiology during working memory and on prefrontal gray matter volume.
Methods
A large sample of healthy subjects was compared for genotypic differences for DRD2 rs1076560 (G>T) and CHNRA5 rs16969968 (G>A) on prefrontal phenotypes, including cognitive performance at the N-Back task, prefrontal physiology with BOLD fMRI during performance of the 2-Back working memory task, and prefrontal morphometry with structural MRI.
Results
We found that DRD2 rs1076560 and CHNRA5 rs16969968 interact to modulate cognitive function, prefrontal physiology during working memory, and prefrontal gray matter volume. More specifically, CHRNA5-AA/DRD2-GT subjects had greater behavioral performance, more efficient prefrontal cortex activity at 2Back working memory task, and greater prefrontal gray matter volume than the other genotype groups.
Conclusions
The present data extend previous studies in animals and enhance our understanding of dopamine and acetylcholine signaling in the human prefrontal cortex, demonstrating interactions elicited by working memory that are modulated by genetic variants in DRD2 and CHRNA5.
doi:10.1371/journal.pone.0095997
PMCID: PMC4018353  PMID: 24819610
2.  DRD2 Genotype-Based Variation of Default Mode Network Activity and of Its Relationship With Striatal DAT Binding 
Schizophrenia Bulletin  2011;39(1):206-216.
The default mode network (DMN) comprises a set of brain regions with “increased” activity during rest relative to cognitive processing. Activity in the DMN is associated with functional connections with the striatum and dopamine (DA) levels in this brain region. A functional single-nucleotide polymorphism within the dopamine D2 receptor gene (DRD2, rs1076560 G > T) shifts splicing of the 2 D2 isoforms, D2 short and D2 long, and has been associated with striatal DA signaling as well as with cognitive processing. However, the effects of this polymorphism on DMN have not been explored. The aim of this study was to evaluate the effects of rs1076560 on DMN and striatal connectivity and on their relationship with striatal DA signaling. Twenty-eight subjects genotyped for rs1076560 underwent functional magnetic resonance imaging during a working memory task and 123 55 I-Fluoropropyl-2-beta-carbomethoxy-3-beta(4-iodophenyl) nortropan Single Photon Emission Computed Tomography ([123I]-FP-CIT SPECT) imaging (a measure of dopamine transporter [DAT] binding). Spatial group-independent component (IC) analysis was used to identify DMN and striatal ICs. Within the anterior DMN IC, GG subjects had relatively greater connectivity in medial prefrontal cortex (MPFC), which was directly correlated with striatal DAT binding. Within the posterior DMN IC, GG subjects had reduced connectivity in posterior cingulate relative to T carriers. Additionally, rs1076560 genotype predicted connectivity differences within a striatal network, and these changes were correlated with connectivity in MPFC and posterior cingulate within the DMN. These results suggest that genetically determined D2 receptor signaling is associated with DMN connectivity and that these changes are correlated with striatal function and presynaptic DA signaling.
doi:10.1093/schbul/sbr128
PMCID: PMC3523900  PMID: 21976709
DRD2; dopamine; default mode network; functional magnetic resonance imaging; single-photon emission computerized tomography
3.  Genetically Determined Measures of Striatal D2 Signaling Predict Prefrontal Activity during Working Memory Performance 
PLoS ONE  2010;5(2):e9348.
Background
Variation of the gene coding for D2 receptors (DRD2) has been associated with risk for schizophrenia and with working memory deficits. A functional intronic SNP (rs1076560) predicts relative expression of the two D2 receptors isoforms, D2S (mainly pre-synaptic) and D2L (mainly post-synaptic). However, the effect of functional genetic variation of DRD2 on striatal dopamine D2 signaling and on its correlation with prefrontal activity during working memory in humans is not known.
Methods
Thirty-seven healthy subjects were genotyped for rs1076560 (G>T) and underwent SPECT with [123I]IBZM (which binds primarily to post-synaptic D2 receptors) and with [123I]FP-CIT (which binds to pre-synaptic dopamine transporters, whose activity and density is also regulated by pre-synaptic D2 receptors), as well as BOLD fMRI during N-Back working memory.
Results
Subjects carrying the T allele (previously associated with reduced D2S expression) had striatal reductions of [123I]IBZM and of [123I]FP-CIT binding. DRD2 genotype also differentially predicted the correlation between striatal dopamine D2 signaling (as identified with factor analysis of the two radiotracers) and activity of the prefrontal cortex during working memory as measured with BOLD fMRI, which was positive in GG subjects and negative in GT.
Conclusions
Our results demonstrate that this functional SNP within DRD2 predicts striatal binding of the two radiotracers to dopamine transporters and D2 receptors as well as the correlation between striatal D2 signaling with prefrontal cortex activity during performance of a working memory task. These data are consistent with the possibility that the balance of excitatory/inhibitory modulation of striatal neurons may also affect striatal outputs in relationship with prefrontal activity during working memory performance within the cortico-striatal-thalamic-cortical pathway.
doi:10.1371/journal.pone.0009348
PMCID: PMC2825256  PMID: 20179754
4.  Dopamine-transporter levels drive striatal responses to apomorphine in Parkinson's disease 
Brain and Behavior  2013;3(3):249-262.
Dopaminergic therapy in Parkinson's disease (PD) can improve some cognitive functions while worsening others. These opposite effects might reflect different levels of residual dopamine in distinct parts of the striatum, although the underlying mechanisms remain poorly understood. We used functional magnetic resonance imaging (fMRI) to address how apomorphine, a potent dopamine agonist, influences brain activity associated with working memory in PD patients with variable levels of nigrostriatal degeneration, as assessed via dopamine-transporter (DAT) scan. Twelve PD patients underwent two fMRI sessions (Off-, On-apomorphine) and one DAT-scan session. Twelve sex-, age-, and education-matched healthy controls underwent one fMRI session. The core fMRI analyses explored: (1) the main effect of group; (2) the main effect of treatment; and (3) linear and nonlinear interactions between treatment and DAT levels. Relative to controls, PD-Off patients showed greater activations within posterior attentional regions (e.g., precuneus). PD-On versus PD-Off patients displayed reduced left superior frontal gyrus activation and enhanced striatal activation during working-memory task. The relation between DAT levels and striatal responses to apomorphine followed an inverted-U-shaped model (i.e., the apomorphine effect on striatal activity in PD patients with intermediate DAT levels was opposite to that observed in PD patients with higher and lower DAT levels). Previous research in PD demonstrated that the nigrostriatal degeneration (tracked via DAT scan) is associated with inverted-U-shaped rearrangements of postsynaptic D2-receptors sensitivity. Hence, it can be hypothesized that individual differences in DAT levels drove striatal responses to apomorphine via D2-receptor-mediated mechanisms.
doi:10.1002/brb3.115
PMCID: PMC3683285  PMID: 23785657
Cognition; DAT; dopamine-agonist; fMRI; Parkinson's disease; working memory
5.  Functional variation of the dopamine D2 receptor gene is associated with emotional control as well as brain activity and connectivity during emotion processing in humans 
Personality traits related to emotion processing are, at least in part, heritable and genetically determined. Dopamine D2 receptor signaling is involved in modulation of emotional behavior and activity of associated brain regions such as the amygdala and the prefrontal cortex. An intronic single nucleotide polymorphism within the D2 receptor gene (DRD2, rs1076560, guanine>thymine - G>T) shifts splicing of the two protein isoforms (D2 short, D2S, mainly presynaptic, and D2 long, D2L) and has been associated with modulation of memory performance and brain activity. Here, our aim was to investigate the association of DRD2 rs1076560 genotype with personality traits of emotional stability and with brain physiology during processing of emotionally relevant stimuli. DRD2 genotype and Big Five Questionnaire scores were evaluated in 134 healthy subjects demonstrating that GG subjects have reduced ‘emotion control’ compared with GT subjects. fMRI in a sample of 24 individuals indicated greater amygdala activity during implicit processing and greater dorsolateral prefrontal cortex (DLPFC) response during explicit processing of facial emotional stimuli in GG subjects compared with GT. Other results also demonstrate an interaction between DRD2 genotype and facial emotional expression on functional connectivity of both amygdala and dorsolateral prefrontal regions with overlapping medial prefrontal areas. Moreover, rs1076560 genotype is associated with differential relationships between amygdala/DLPFC functional connectivity and emotion control scores. These results suggest that genetically determined D2 signaling may explain part of personality traits related to emotion processing and individual variability in specific brain responses to emotionally relevant inputs.
doi:10.1523/JNEUROSCI.3609-09.2009
PMCID: PMC2834475  PMID: 19940176
amygdala; DRD2; dopamine; emotion; fMRI; prefrontal cortex
6.  Functional variants of the dopamine receptor D2 gene modulate prefronto-striatal phenotypes in schizophrenia 
Brain  2008;132(2):417-425.
Dopamine D2 receptor signalling is strongly implicated in the aetiology of schizophrenia. We have recently characterized the function of three DRD2 SNPs: rs12364283 in the promoter affecting total D2 mRNA expression; rs2283265 and rs1076560, respectively in introns 5 and 6, shifting mRNA splicing to two functionally distinct isoforms, the short form of D2 (D2S) and the long form (D2L). These two isoforms differentially contribute to dopamine signalling in prefrontal cortex and in striatum. We performed a case–control study to determine association of these variants and of their main haplotypes with several schizophrenia-related phenotypes. We demonstrate that the minor allele in the intronic variants is associated with reduced expression of %D2S of total mRNA in post-mortem prefrontal cortex, and with impaired working memory behavioural performance, both in patients and controls. However, the fMRI results show opposite effects in patients compared with controls: enhanced engagement of prefronto-striatal pathways in controls and reduced activity in patients. Moreover, the promoter variant is also associated with working memory activity in prefrontal cortex and striatum of patients, and less robustly with negative symptoms scores. Main haplotypes formed by the three DRD2 variants showed significant associations with these phenotypes consistent with those of the individual SNPs. Our results indicate that the three functional DRD2 variants modulate schizophrenia phenotypes possibly by modifying D2S/D2L ratios in the context of different total D2 density.
doi:10.1093/brain/awn248
PMCID: PMC2640212  PMID: 18829695
dopamine; D2 receptor; working memory; prefrontal cortex; striatum
7.  Prefronto-striatal physiology is associated with schizotypy and is modulated by a functional variant of DRD2 
“Schizotypy” is a latent organization of personality related to the genetic risk for schizophrenia. Some evidence suggests that schizophrenia and schizotypy share some biological features, including a link to dopaminergic D2 receptor signaling. A polymorphism in the D2 gene (DRD2 rs1076560, guanine > thymine (G > T)) has been associated with the D2 short/long isoform expression ratio, as well as striatal dopamine signaling and prefrontal cortical activity during different cognitive operations, which are measures that are altered in patients with schizophrenia. Our aim is to determine the association of schizotypy scores with the DRD2 rs1076560 genotype in healthy individuals and their interaction with prefrontal activity during attention and D2 striatal signaling. A total of 83 healthy subjects were genotyped for DRD2 rs1076560 and completed the Schizotypal Personality Questionnaire (SPQ). Twenty-six participants underwent SPECT with [123I]IBZM D2 receptor radiotracer, while 68 performed an attentional control task during fMRI. We found that rs1076560 GT subjects had greater SPQ scores than GG individuals. Moreover, the interaction between schizotypy and the GT genotype predicted prefrontal activity and related attentional behavior, as well as striatal binding of IBZM. No interaction was found in GG individuals. These results suggest that rs1076560 GT healthy individuals are prone to higher levels of schizotypy, and that the interaction between rs1076560 and schizotypy scores modulates phenotypes related to the pathophysiology of schizophrenia, such as prefrontal activity and striatal dopamine signaling. These results provide systems-level qualitative evidence for mapping the construct of schizotypy in healthy individuals onto the schizophrenia continuum.
doi:10.3389/fnbeh.2014.00235
PMCID: PMC4089730  PMID: 25071490
schizotypy; dopamine; DRD2; fMRI; SPECT
8.  ADHD-Derived Coding Variation in the Dopamine Transporter Disrupts Microdomain Targeting and Trafficking Regulation 
The Journal of Neuroscience  2012;32(16):5385-5397.
Attention-Deficit Hyperactivity Disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain imaging studies suggest a contribution of altered dopamine (DA) signaling in ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at pre- and post-synaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified a human DAT (hDAT) coding variant, R615C, located in the transporter’s distal C-terminus, a region previously implicated in constitutive and regulated transporter trafficking. Here we demonstrate that whereas wildtype DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively, and demonstrate insensitivity to the endocytic effects of AMPH and protein kinase C (PKC) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered calcium/calmodulin-dependent protein kinase II (CaMKII) and flotillin-1 interactions. Using C-terminal peptides derived from wildtype DAT and the R615C variant, we establish that the DAT 615C C-terminus can act dominantly to preclude AMPH regulation of wildtype DAT. Mutagenesis of DAT C-terminal sequences suggest that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies support a coupling of DAT microdomain localization with transporter regulation and provide evidence of perturbed DAT activity and DA signaling as a risk determinant for ADHD.
doi:10.1523/JNEUROSCI.6033-11.2012
PMCID: PMC3342037  PMID: 22514303
ADHD; dopamine; transporter; amphetamine; PKC; trafficking
9.  Comparison of positron emission tomography, cognition, and brain volume in Alzheimer's disease with and without severe abnormalities of white matter. 
OBJECTIVES--To examine cerebral metabolism, cognitive performance, and brain volumes in healthy controls and two groups of patients with probable Alzheimer's disease, one group with severe abnormalities of white matter (DAT+) and the other group with none, or minimal abnormalities (DAT-). METHODS--Neuropsychological tests, CT, MRI, quantitative MRI, and PET studies were carried out to allow comparison between the DAT+ and DAT- groups and the healthy controls. RESULTS--Compared with the healthy controls, both demented groups had significantly reduced global and regional cerebral metabolism, significant brain atrophy, and significantly lower scores on neuropsychological testing. The DAT- patient group showed a pattern of parietal-temporal cerebral metabolic reductions and neuropsychological performance deficits typical of Alzheimer's disease. In addition, metabolism in the association neocortex (AD ratio) and measures of neuropsychological task performance were significantly correlated in the DAT- patient group. Comparison of DAT+ with DAT- patients showed a significantly higher ratio of parietal to whole brain glucose utilisation for the DAT+ group. Moreover, when comparing group z score differences from the healthy controls, the DAT+ group had, on average, smaller differences from controls in the frontal, parietal, and temporal regions than did the DAT- group. Discriminant analysis using metabolic ratios of the frontal, parietal, and temporal regions showed cerebral metabolic patterns to be significantly different among the DAT+, the DAT-, and the healthy controls. These differences were due primarily to relatively higher frontal, parietal, and temporal metabolic ratios in the DAT+ group which resulted in discriminant scores for the DAT+ group between the healthy controls and the DAT- group. Group mean scores on tests of neuropsychological performance were not significantly different between the DAT- and DAT+ patients. By contrast with the DAT- group, however, no significant correlations between the AD ratio and any neuropsychological task were seen in the DAT+ group. Multiple regression analysis showed significant between group differences in the relation between the AD ratio and neuropsychological scores on three tasks. The slopes of the relations between the AD ratio and memory scores (memory and freedom from distractability deviation quotient of the Wechsler adult intelligence scale (WMDQ)) also were significantly different for the two groups. CONCLUSIONS--Although multiple causes for abnormalities of white matter exist in patients with Alzheimer's disease, these data suggest that the presence of severe abnormalities of white matter indicate a second pathological process in the DAT+ patients. The DAT- patients showed the parietal-temporal metabolic deficits and correlations between association neocortical metabolism and neuropsychological task performance typical of patients with Alzheimer's disease. By contrast, the DAT+ group had a pattern of cerebral metabolism significantly different from healthy controls and DAT+ patients, as well as no significant correlations between metabolism in the association neocortex and neuropsychological performance. These differences probably reflect the superimposed pathology of the abnormalities of white matter which may exert their affect through disruption of long corticocortical pathways.
Images
PMCID: PMC1073796  PMID: 8708645
10.  Dissociable fronto-striatal effects of dopamine D2 receptor stimulation on cognitive vs. motor flexibility 
Genetic and pharmacological studies suggest an important role of the dopamine D2 receptor (DRD2) in flexible behavioral adaptation, mostly shown in reward-based learning paradigms. Recent evidence from imaging genetics indicates that also intentional cognitive flexibility, associated with lateral frontal cortex, is affected by variations in DRD2 signaling. In the present functional MRI study, we tested the effects of a direct pharmacological manipulation of DRD2 stimulation on intentional flexibility in a task-switching context, requiring switches between cognitive task rules and between response hands. In a double-blind, counterbalanced design, participants received either a low dose of the DRD2 agonist bromocriptine or a placebo in two separate sessions. Bromocriptine modulated BOLD signal during rule switching: rule-switching-related activity in the left posterior lateral frontal cortex and in the striatum was increased compared to placebo, at comparable performance levels. Fronto-striatal connectivity under bromocriptine was slightly increased for rule switches compared to rule repetitions. Hand-switching-related activity, in contrast, was reduced under bromocriptine in sensori-motor regions. Our results provide converging evidence for an involvement of DRD2 signaling in fronto-striatal mechanisms underlying intentional flexibility, and indicate that the neural mechanisms underlying different types of flexibility (cognitive vs. motor) are affected differently by increased dopaminergic stimulation.
doi:10.1016/j.cortex.2013.04.002
PMCID: PMC3795948  PMID: 23660437
bromocriptine; psychopharmacology; intentional flexibility; functional magnetic resonance imaging (fMRI)
11.  Dopamine Transporters in Striatum Correlate with Deactivation in the Default Mode Network during Visuospatial Attention 
PLoS ONE  2009;4(6):e6102.
Background
Dopamine and dopamine transporters (DAT, which regulate extracellular dopamine in the brain) are implicated in the modulation of attention but their specific roles are not well understood. Here we hypothesized that dopamine modulates attention by facilitation of brain deactivation in the default mode network (DMN). Thus, higher striatal DAT levels, which would result in an enhanced clearance of dopamine and hence weaker dopamine signals, would be associated to lower deactivation in the DMN during an attention task.
Methodology/Principal Findings
For this purpose we assessed the relationship between DAT in striatum (measured with positron emission tomography and [11C]cocaine used as DAT radiotracer) and brain activation and deactivation during a parametric visual attention task (measured with blood oxygenation level dependent functional magnetic resonance imaging) in healthy controls. We show that DAT availability in caudate and putamen had a negative correlation with deactivation in ventral parietal regions of the DMN (precuneus, BA 7) and a positive correlation with deactivation in a small region in the ventral anterior cingulate gyrus (BA 24/32). With increasing attentional load, DAT in caudate showed a negative correlation with load-related deactivation increases in precuneus.
Conclusions/Significance
These findings provide evidence that dopamine transporters modulate neural activity in the DMN and anterior cingulate gyrus during visuospatial attention. Our findings suggest that dopamine modulates attention in part by regulating neuronal activity in posterior parietal cortex including precuneus (region involved in alertness) and cingulate gyrus (region deactivated in proportion to emotional interference). These findings suggest that the beneficial effects of stimulant medications (increase dopamine by blocking DAT) in inattention reflect in part their ability to facilitate the deactivation of the DMN.
doi:10.1371/journal.pone.0006102
PMCID: PMC2699543  PMID: 19564918
12.  COMT and ANKK1-Taq-Ia Genetic Polymorphisms Influence Visual Working Memory 
PLoS ONE  2013;8(1):e55862.
Complex cognitive tasks such as visual working memory (WM) involve networks of interacting brain regions. Several neurotransmitters, including an appropriate dopamine concentration, are important for WM performance. A number of gene polymorphisms are associated with individual differences in cognitive task performance. COMT, for example, encodes catechol-o-methyl transferase the enzyme primarily responsible for catabolizing dopamine in the prefrontal cortex. Striatal dopamine function, linked with cognitive tasks as well as habit learning, is influenced by the Taq-Ia polymorphism of the DRD2/ANKK1 gene complex; this gene influences the density of dopamine receptors in the striatum. Here, we investigated the effects of these polymorphisms on a WM task requiring the maintenance of 4 or 6 items over delay durations of 1 or 5 seconds. We explored main effects and interactions between the COMT and DRD2/ANKK1-Taq-Ia polymorphisms on WM performance. Participants were genotyped for COMT (Val158Met) and DRD2/ANKK1-Taq-Ia (A1+, A1−) polymorphisms. There was a significant main effect of both polymorphisms. Participants' WM reaction times slowed with increased Val loading such that the Val/Val homozygotes made the slowest responses and the Met/Met homozygotes were the fastest. Similarly, WM reaction times were slower and more variable for the DRD2/ANKK1-Taq-Ia A1+ group than the A1− group. The main effect of COMT was only apparent in the DRD2/ANKK1-Taq-Ia A1− group. These findings link WM performance with slower dopaminergic metabolism in the prefrontal cortex as well as a greater density of dopamine receptors in the striatum.
doi:10.1371/journal.pone.0055862
PMCID: PMC3561341  PMID: 23383291
13.  Reduced D2-mediated signaling activity and trans-synaptic upregulation of D1 and D2 dopamine receptors in mice overexpressing the dopamine transporter 
Cellular signalling  2008;21(1):87-94.
The dopamine transporter (DAT) regulates the temporal and spatial actions of dopamine by reuptaking this neurotransmitter into the presynaptic neurons. We recently generated transgenic mice overexpressing DAT (DAT-tg) that have a 3-fold increase in DAT protein levels which results in a 40% reduction of the extracellular DA concentration in the striatum. The aim of this study was to examine the effect of this reduction in dopaminergic tone on postsynaptic responses mediated by dopamine receptors. We report here that DAT-tg mice have increased levels of striatal D1 (30%) and D2 (~60%) dopamine receptors with D1 receptor signaling components not significantly altered as evidenced by unaffected basal or stimulated levels of phospho-GluR1 (Ser845) and phospho-ERK2. However, the novel D2 mediated Akt signaling is markedly altered in DAT-tg animals. In particular, there is a 300% increase in the basal levels of phospho-Akt in the striatum of DAT-tg, reflecting the reduced extracellular dopamine tone in these animals. This increase in basal pAkt levels can be pharmacologically recapitulated by partial dopamine depletion in WT mice treated with the selective tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (α-MPT). Behaviorally, DAT-tg animals demonstrate an augmented synergistic interaction between up-regulated D1 and D2 receptors which results in increased climbing behavior in transgenic mice after stimulation with either apomorphine or a co-administration of selective D1 and D2 receptor agonists. In sum, our study reveals that hypodopaminegia caused by up-regulation of DAT results in significant alterations at postsynaptic receptor function with most notable dysregulation at the level of D2 receptor signaling.
doi:10.1016/j.cellsig.2008.09.011
PMCID: PMC3536494  PMID: 18929645
Dopamine transporter (DAT); BAC Transgenic mice; hypodopaminergia; dopamine receptors; Akt; apomorphine
14.  Interacting Effects of Naltrexone and OPRM1 and DAT1 Variation on the Neural Response to Alcohol Cues 
Neuropsychopharmacology  2012;38(3):414-422.
Variation at a single nucleotide polymorphism in the μ-opioid receptor gene (OPRM1), A118G (Asn40Asp), may moderate naltrexone (NTX) effects in alcohol dependence. Both NTX and A118G variation have also been reported to affect alcohol cue-elicited brain activation. This study investigated whether sub-acute NTX treatment and A118G genotype interacted in their effects on cue-elicited activation of the ventral striatum (VS), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC). Secondarily, variation at a variable number tandem repeat polymorphism in the dopamine transporter gene (DAT1/SLC6A3), which has been associated with increased reward-related activation in VS, was analyzed as a moderator of medication and A118G effects. Seventy-four non-treatment-seeking alcohol-dependent individuals, half preselected to carry at least one copy of the A118G G (Asp) allele, were randomized to NTX (50 mg) or placebo for 7 days, and performed an fMRI alcohol cue reactivity task on day 6. Region-of-interest analyses indicated no main effects of medication or A118G genotype. However, these factors interacted in their effects on OFC activation, such that, among NTX-treated individuals, G-allele carriers had less activation than A-allele homozygotes. DAT1 variation also moderated medication/A118G effects. There was a three-way interaction between medication and A118G and DAT1 genotypes on VS activation, such that, among G-allele carriers who received NTX, DAT1 10-repeat-allele (10R) homozygotes had less activation than 9-repeat-allele (9R) carriers. Further, 10R homozygotes who received NTX had less mPFC activation than 9R carriers. Polymorphic variation in OPRM1 and DAT1 should be considered in future studies of NTX, particularly regarding its effects on reward processing.
doi:10.1038/npp.2012.195
PMCID: PMC3547192  PMID: 23032071
alcoholism; opioid antagonist; functional neuroimaging; SNP; VNTR; Alcohol & Alcoholism; Clinical Pharmacology/Clinical Trials; fMRI; Imaging; Clinical or Preclinical; Naltrexone; opioid antagonist; Pharmacogenetics / Pharmacogenomics; SNP
15.  Lobeline Attenuates Neonatal Ethanol-Mediated Changes in Hyperactivity and Dopamine Transporter Function in the Prefrontal Cortex in Rats 
Neuroscience  2011;206:245-254.
Current therapies for attention deficit hyperactivity disorder (ADHD) have varying efficacy in individuals with fetal alcohol spectrum disorders (FASD), suggesting that alternative therapeutics are needed. Developmental exposure to ethanol produces changes in dopamine (DA) systems, and DA has also been implicated in ADHD pathology. In the current study, lobeline, which interacts with proteins in dopaminergic presynaptic terminals, was evaluated for its ability to attenuate neonatal ethanol-induced locomotor hyperactivity and alterations in dopamine transporter (DAT) function in striatum and prefrontal cortex (PFC). From postnatal days (PND) 1–7, male and female rat pups were intubated twice daily with either 3 g/kg ethanol or milk, or were not intubated (non-intubated control) as a model for “third trimester” ethanol exposure. On PND 21 and 22, pups received acute lobe-line (0, 0.3, 1, or 3 mg/kg), and locomotor activity was assessed. On PND 23–25, pups again received an acute injection of lobeline (1 or 3 mg/kg), and DAT kinetic parameters (Km and Vmax) were determined. Results demonstrated that neonatal ethanol produced locomotor hyperactivity on PND 21 that was reversed by lobeline (1 and 3 mg/kg). Although striatal DAT function was not altered by neonatal ethanol or acute lobeline, neonatal ethanol exposure increased the Vmax for DAT in the PFC, suggesting an increase in DAT function in PFC. Lobeline ameliorated this effect on PFC Vmax at the same doses that decreased hyperactivity. Methylphenidate, the gold standard therapeutic for ADHD, was also evaluated for comparison with lobeline. Methylphenidate decreased DAT Vmax and Km in PFC from ethanol-treated pups. Thus, lobeline and methylphenidate differentially altered DAT function following neonatal ethanol exposure. Collectively, these findings provide support that lobeline may be a useful phar-macotherapy for some of the deficits associated with neonatal ethanol exposure.
doi:10.1016/j.neuroscience.2011.11.018
PMCID: PMC3996807  PMID: 22119644
ethanol; dopamine transporter; prefrontal cortex; lobeline; hyperactivity; preadolescent
16.  Single and Binge Methamphetamine Administrations Have Different Effects on the Levels of Dopamine D2 Autoreceptor and Dopamine Transporter in Rat Striatum 
Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum. Dopamine D2 receptor antagonists and dopamine transporter (DAT) inhibitors protect against neurotoxicity of the drug by decreasing intracellular dopamine content and, consequently, dopamine autoxidation and production of reactive oxygen species. In vitro, amphetamines regulate D2 receptor and DAT functions via regulation of their intracellular trafficking. No data exists on axonal transport of both proteins and there is limited data on their interactions in vivo. The aim of the present investigation was to examine synaptosomal levels of presynaptic D2 autoreceptor and DAT after two different regimens of METH and to determine whether METH affects the D2 autoreceptor-DAT interaction in the rat striatum. We found that, as compared to saline controls, administration of single high-dose METH decreased D2 autoreceptor immunoreactivity and increased DAT immunoreactivity in rat striatal synaptosomes whereas binge high-dose METH increased immunoreactivity of D2 autoreceptor and had no effect on DAT immunoreactivity. Single METH had no effect on D2 autoreceptor-DAT interaction whereas binge METH increased the interaction between the two proteins in the striatum. Our results suggest that METH can affect axonal transport of both the D2 autoreceptor and DAT in an interaction-dependent and -independent manner.
doi:10.3390/ijms15045884
PMCID: PMC4013602  PMID: 24717411
dopamine D2 receptor; dopamine transporter; methamphetamine; striatum; in vivo
17.  Dopamine transporter DAT and receptor DRD2 variants affect risk of lethal cocaine abuse: a gene–gene–environment interaction 
Translational Psychiatry  2013;3(1):e222-.
Epistatic gene–gene interactions could contribute to the heritability of complex multigenic disorders, but few examples have been reported. Here, we focus on the role of aberrant dopaminergic signaling, involving the dopamine transporter DAT, a cocaine target, and the dopamine D2 receptor, which physically interacts with DAT. Splicing polymorphism rs2283265 of DRD2, encoding D2 receptors, were shown to confer risk of cocaine overdose/death (odds ratio ∼3) in subjects and controls from the Miami Dade County Brain Bank.1 Risk of cocaine-related death attributable to the minor allele of rs2283265 was significantly enhanced to OR=7.5 (P=0.0008) in homozygous carriers of the main 6-repeat allele of DAT rs3836790, a regulatory VNTR in intron8 lacking significant effect itself. In contrast, carriers of the minor 5-repeat DAT allele showed no significant risk (OR=1.1, P=0.84). DAT rs3836790 and DRD2 rs2283265 also interacted by modulating DAT protein activity in the ventral putamen of cocaine abusers. In high-linkage disequilibrium with the VNTR, DAT rs6347 in exon9 yielded similar results. Assessing the impact of DAT alone, a rare DAT haplotype formed by the minor alleles of rs3836790 and rs27072, a regulatory DAT variant in the 3′-UTR, occurred in nearly one-third of the cocaine abusers but was absent in African American controls, apparently conferring strong risk. These results demonstrate gene–gene–drug interaction affecting risk of fatal cocaine intoxication.
doi:10.1038/tp.2012.146
PMCID: PMC3566726  PMID: 23340505
cocaine-related death; DAT; DRD2; epistasis; gene–gene interaction; gene regulation; haplotype
18.  Dopamine dynamics associated with, and resulting from, schedule-induced alcohol self-administration: Analyses in dopamine transporter knockout mice 
Alcohol (Fayetteville, N.Y.)  2011;45(4):325-339.
Preclinical and clinical evidence suggest an association between alcoholism and the primary regulator of extracellular dopamine concentrations, the dopamine transporter (DAT). However, the nature of this association is unclear. We determined if ten days of voluntary alcohol self-administration followed by withdrawal could directly alter DAT function, or if genetically-mediated changes in DAT function and/or availability could influence vulnerability to alcohol abuse. Heterozygous (DAT+/-) and homozygous mutant (DAT-/-) and wildtype (DAT+/+) mice were allowed to consume 5% alcohol in a schedule-induced polydipsia (SIP) task. In vivo fixed potential amperometry in anesthetized mice was used to (1) identify functional characteristics of mesoaccumbens dopamine neurons related to genotype, including dopamine autoreceptor (DAR) sensitivity, DAT efficiency, and DAT capacity, (2) determine if any of these characteristics correlated with alcohol drinking observed in DAT+/+ and DAT+/- animals, and (3) determine if SIP-alcohol self-administration altered DAR sensitivity, DAT efficiency, and DAT capacity by comparing these characteristics in wildtype (DAT+/+) mice that were SIP-alcohol naïve, with those that had undergone SIP-alcohol testing. DAT-/- mice consumed significantly less alcohol during testing and this behavioral difference was related to significant differences in DAR sensitivity, DAT efficiency, and DAT capacity. These functional characteristics were correlated to varying degrees with g/kg alcohol consumption in DAT+/+ and DAT+/- mice. DAR sensitivity was consistently reduced and DAT efficiency was enhanced in SIP-alcohol experienced DAT+/+ mice in comparison to naïve animals. These results indicate that DAR sensitivity is reduced by SIP-alcohol consumption and that DAT efficiency is modified by genotype as well as SIP-alcohol exposure. DAT capacity appeared to be strictly associated with SIP-alcohol consumption.
doi:10.1016/j.alcohol.2010.12.006
PMCID: PMC3095708  PMID: 21354763
dopamine transporter; dopamine autoreceptor; schedule-induced polydipsia; fixed potential amperometry; alcohol self-administration
19.  Modulation by Cocaine of Dopamine Receptors through miRNA-133b in Zebrafish Embryos 
PLoS ONE  2012;7(12):e52701.
The use of cocaine during pregnancy can affect the mother and indirectly might alter the development of the embryo/foetus. Accordingly, in the present work our aim was to study in vivo (in zebrafish embryos) the effects of cocaine on the expression of dopamine receptors and on miR-133b. These embryos were exposed to cocaine hydrochloride (HCl) at 5 hours post-fertilization (hpf) and were then collected at 8, 16, 24, 48 and 72 hpf to study the expression of dopamine receptors, drd1, drd2a, drd2b and drd3, by quantitative real time PCR (qPCR) and in situ hybridization (ISH, only at 24 hpf). Our results indicate that cocaine alters the expression of the genes studied, depending on the stage of the developing embryo and the type of dopamine receptor. We found that cocaine reduced the expression of miR-133b at 24 and 48 hpf in the central nervous system (CNS) and at the periphery by qPCR and also that the spatial distribution of miR-133b was mainly seen in somites, a finding that suggests the involvement of miR-133b in the development of the skeletal muscle. In contrast, at the level of the CNS miR-133b had a weak and moderate expression at 24 and 48 hpf. We also analysed the interaction of miR-133b with the Pitx3 and Pitx3 target genes drd2a and drd2b, tyrosine hydroxylase (th) and dopamine transporter (dat) by microinjection of the Pitx3-3'UTR sequence. Microinjection of Pitx3-3'UTR affected the expression of pitx3, drd2a, drd2b, th and dat. In conclusion, in the present work we describe a possible mechanism to account for cocaine activity by controlling miR-133b transcription in zebrafish. Via miR-133b cocaine would modulate the expression of pitx3 and subsequently of dopamine receptors, dat and th. These results indicate that miRNAs can play an important role during embryogenesis and in drug addiction.
doi:10.1371/journal.pone.0052701
PMCID: PMC3528707  PMID: 23285158
20.  Immunization with DAT fragments is associated with long-term striatal impairment, hyperactivity and reduced cognitive flexibility in mice 
Background
Possible interactions between nervous and immune systems in neuro-psychiatric disorders remain elusive. Levels of brain dopamine transporter (DAT) have been implicated in several impulse-control disorders, like attention deficit / hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD). Here, we assessed the interplay between DAT auto-immunity and behavioural / neurochemical phenotype.
Methods
Male CD-1 mice were immunized with DAT peptide fragments (DAT-i), or vehicle alone (VEH), to generate elevated circulating levels of DAT auto-antibodies (aAbs). Using an operant delay-of-reward task (20 min daily sessions; timeout 25 sec), mice had a choice between either an immediate small amount of food (SS), or a larger amount of food after a delay (LL), which increased progressively across sessions (from 0 to 150 sec).
Results
DAT-i mice exhibited spontaneous hyperactivity (2 h-longer wake-up peak; a wake-up attempt during rest). Two sub-populations differing in behavioural flexibility were identified in the VEH control group: they showed either a clear-cut decision to select LL or clear-cut shifting towards SS, as expected. Compared to VEH controls, choice-behaviour profile of DAT-i mice was markedly disturbed, together with long-lasting alterations of the striatal monoamines. Enhanced levels of DA metabolite HVA in DAT-i mice came along with slower acquisition of basal preferences and with impaired shifting; elevation also in DOPAC levels was associated with incapacity to change a rigid selection strategy. This scarce flexibility of performance is indicative of a poor adaptation to task contingencies.
Conclusions
Hyperactivity and reduced cognitive flexibility are patterns of behaviour consistent with enduring functional impairment of striatal regions. It is yet unclear how anti-DAT antibodies could enter or otherwise affect these brain areas, and which alterations in DAT activity exactly occurred after immunization. Present neuro-behavioural alterations, coming along with an experimentally-induced rise of circulating DAT-directed aAbs, open the issue of a potential role for auto-immunity in vulnerability to impulse-control disorders.
doi:10.1186/1744-9081-8-54
PMCID: PMC3537576  PMID: 23192105
Auto-antibodies to neuro-receptors; DAT; Delay of reward; Flexibility of choice behaviour; ADHD; OCD
21.  Neural response to working memory load varies by dopamine transporter genotype in children 
NeuroImage  2010;53(3):970-977.
Inheriting two (10/10) relative to one (9/10) copy of the 10-repeat allele of the dopamine transporter genotype (DAT1) is associated with Attention Deficit Hyperactivity Disorder, a childhood disorder marked by poor executive function. We examined whether functional anatomy underlying working memory, a component process of executive function, differed by DAT1 in 7-12 year-old typically developing children. 10/10 and 9/10 carriers performed a verbal n-back task in two functional magnetic resonance imaging (fMRI) runs varying in working memory load, high (2-back vs. 1-back) and low (1-back vs. 0-back). Performance accuracy was superior in 9/10 than 10/10 carriers in the high but not low load runs. Examination of each run separately revealed that frontal-striatal-parietal regions were more activated in 9/10 than 10/10 carriers in the high load run; the groups did not differ in the low load run. Examination of load effects revealed a DAT1 X Load interaction in the right hemisphere in the caudate, our a priori region of interest. Exploratory analysis at a more liberal threshold revealed this interaction in other basal ganglia regions (putamen, and substantial nigra/subthalamic nuclei – SN/STN) and in medial parietal cortex (left precuneus). The striatal and parietal regions were more activated in 9/10 carriers under high than low load, and DAT1 differences (9/10 > 10/10) were evident only under high load. In contrast, SN/STN tended to be more activated in 10/10 carriers under low than high load and DAT1 differences (10/10 > 9/10) were evident only under low load. Thus, 10-repeat homozygosity of DAT1 was associated with reduced performance and a lack of increased basal ganglia involvement under higher working memory demands.
doi:10.1016/j.neuroimage.2009.12.104
PMCID: PMC2888738  PMID: 20053379
caudate; fMRI; N-back; functional polymorphism; DAT1; executive function
22.  Relationship of DAT1 and adult ADHD to task-positive and task-negative working memory networks 
Psychiatry research  2011;193(1):7-16.
Alterations in working memory, default-mode network (DMN), and dopamine transporter have all been proposed as endophenotypes for Attention-Deficit Hyperactivity/Disorder (ADHD). Despite evidence that these systems are interrelated, their relationship to each other has never been studied in the context of ADHD. In order to understand the potential mediating effects of task-positive and task-negative networks between DAT1 and diagnosis, we tested effects of genotype and diagnosis on regions of positive and negative BOLD signal change (as measured with fMRI) in 53 adults with ADHD and 38 control subjects during a working memory task. We also examined the relationship of these responses to ADHD symptoms. Our results yielded four principal findings: 1) association of 9R with adult ADHD, 2) marginal DAT1 association with task-related suppression in left medial PFC, 3) marginal genotype × diagnosis interaction in the dorsal anterior cingulate cortex, and 4) correlation of DMN suppression to ADHD symptoms. These findings replicate the association of the 9R allele with adult ADHD. Further, we show that DMN suppression is likely linked to DAT1 and to severity of inattention in ADHD. DMN may therefore be a target of DAT1 effects, and lie on the path between the gene and inattention in ADHD.
doi:10.1016/j.pscychresns.2011.01.006
PMCID: PMC3105199  PMID: 21596533
fMRI; default-mode network; attention
23.  Protein kinase Cβ is a modulator of the dopamine D2 autoreceptor-activated trafficking of the dopamine transporter 
Journal of neurochemistry  2013;125(5):663-672.
The strength and duration of extracellular dopamine concentrations are regulated by the presynaptic dopamine transporter (DAT) and dopamine D2 autoreceptors (D2autoRs). There is a functional interaction between these two proteins. Activation of D2autoRs increases DAT trafficking to the surface whereas disruption of this interaction compromises activities of both proteins and alters dopaminergic transmission. Previously we reported that DAT expression and activity are subject to modulation by protein kinase Cβ (PKCβ). Here, we further demonstrate that PKCβ is integral for the interaction between DAT and D2autoR. Inhibition or absence of PKCβ abolished the communication between DAT and D2autoR. In mouse striatal synaptosomes and transfected N2A cells, the D2autoR-stimulated membrane insertion of DAT was abolished by PKCβ inhibition. Moreover, D2autoR-stimulated DAT trafficking is mediated by a PKCβ-ERK (extracellular signal-regulated kinase) signaling cascade where PKCβ is upstream of ERK. The increased surface DAT expression upon D2autoR activation resulted from enhanced DAT recycling as opposed to reduced internalization. Further, PKCβ promoted accelerated DAT recycling. Our study demonstrates that PKCβ critically regulates D2autoR-activated DAT trafficking and dopaminergic signaling. PKCβ is a potential drug target for correcting abnormal extracellular dopamine levels in diseases such as drug addiction and schizophrenia.
doi:10.1111/jnc.12229
PMCID: PMC3660534  PMID: 23458603
dopamine transporter; dopamine D2 autoreceptor; Protein kinase Cβ; trafficking; extracellular signal regulated protein kinase
24.  Regulation of dopamine transporter activity by carboxypeptidase E 
Molecular Brain  2009;2:10.
Background
The dopamine transporter (DAT) plays a critical role in terminating the action of dopamine by rapid reuptake into the presynaptic neuron. Previous studies have revealed that the DAT carboxyl terminus (DAT-CT) can directly interact with other cellular proteins and regulate DAT function and trafficking.
Results
Here, we have identified that carboxypeptidase E (CPE), a prohormone processing exopeptidase and sorting receptor for the regulated secretory pathway, interacts with the DAT-CT and affects DAT function. Mammalian cell lines coexpressing CPE and DAT exhibited increased DAT-mediated dopamine uptake activity compared to cells expressing DAT alone. Moreover, coexpression of an interfering DAT-CT minigene inhibited the effects of CPE on DAT. Functional changes caused by CPE could be attributed to enhanced DAT expression and subsequent increase in DAT cell surface localization, due to decreased DAT degradation. In addition, CPE association could reduce the phosphorylation state of DAT on serine residues, potentially leading to reduced internalization, thus stabilizing plasmalemmal DAT localization.
Conclusion
Taken together, our results reveal a novel role for CPE in the regulation of DAT trafficking and DAT-mediated DA uptake, which may provide a novel target in the treatment of dopamine-governed diseases such as drug addiction and obesity.
doi:10.1186/1756-6606-2-10
PMCID: PMC2687442  PMID: 19419578
25.  Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain 
PLoS ONE  2010;5(7):e11506.
The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn2+ into the prefrontal cortex indicated that DAT KO mice have a truncated Mn2+ distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn2+ transport into more posterior midbrain nuclei and contralateral mesolimbic structures at 26 hr post-injection. Thus, DAT KO mice appear, at this level of anatomic resolution, to have preserved cortico-striatal-thalamic connectivity but diminished robustness of reward-modulating circuitry distal to the thalamus. This is in contradistinction to the state of this circuitry in serotonin transporter KO mice where we observed more robust connectivity in more posterior brain regions using methods identical to those employed here.
doi:10.1371/journal.pone.0011506
PMCID: PMC2901340  PMID: 20634895

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