40 results on '"Gu, Howard H."'
Search Results
2. Abolished Cocaine Reward in Mice with a Cocaine-Insensitive Dopamine Transporter
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Chen, Rong, Tilley, Michael R., Wei, Hua, Zhou, Fuwen, Zhou, Fu-Ming, Ching, San, Quan, Ning, Stephens, Robert L., Hill, Erik R., Nottoli, Timothy, Han, Dawn D., and Gu, Howard H.
- Published
- 2006
- Full Text
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3. Risk-attitude selection bias in subject pools for experiments involving neuroimaging and blood samples
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Roe, Brian E., Haab, Timothy C., Beversdorf, David Q., Gu, Howard H., and Tilley, Michael R.
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- 2009
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4. Restoration of cocaine stimulation and reward by reintroducing wild type dopamine transporter in adult knock-in mice with a cocaine-insensitive dopamine transporter
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Wu, Haiyin, OʼNeill, Brian, Han, Dawn D., Thirtamara-Rajamani, Keerthi, Wang, Yanlin, and Gu, Howard H.
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- 2014
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5. Behavior of knock-in mice with a cocaine-insensitive dopamine transporter after virogenetic restoration of cocaine sensitivity in the striatum
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OʼNeill, Brian, Tilley, Michael R., Han, Dawn D., Thirtamara-Rajamani, Keerthi, Hill, Erik R., Bishop, Georgia A., Zhou, Fu-Ming, During, Matthew J., and Gu, Howard H.
- Published
- 2014
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- View/download PDF
6. Direct evidence that two cysteines in the dopamine transporter form a disulfide bond
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Chen, Rong, Wei, Hua, Hill, Erik R., Chen, Lucy, Jiang, Liying, Han, Dawn D., and Gu, Howard H.
- Published
- 2007
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7. Amphetamine-induced locomotion in a hyperdopaminergic ADHD mouse model depends on genetic background
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OʼNeill, Brian and Gu, Howard H.
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- 2013
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8. Loss of striatal cannabinoid CB1 receptor function in attention-deficit / hyperactivity disorder mice with point-mutation of the dopamine transporter
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Castelli, Maura, Federici, Mauro, Rossi, Silvia, De Chiara, Valentina, Napolitano, Francesco, Studer, Valeria, Motta, Caterina, Sacchetti, Lucia, Romano, Rosaria, Musella, Alessandra, Bernardi, Giorgio, Siracusano, Alberto, Gu, Howard H., Mercuri, Nicola B., Usiello, Alessandro, and Centonze, Diego
- Published
- 2011
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9. A triple mutation in the second transmembrane domain of mouse dopamine transporter markedly decreases sensitivity to cocaine and methylphenidate
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Chen, Rong, Han, Dawn D., and Gu, Howard H.
- Published
- 2005
10. Cocaine reward and locomotion stimulation in mice with reduced dopamine transporter expression
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Tiao Narry, Han Dawn D, Zhuang Xiaoxi, Cagniard Barbara, Tilley Michael R, and Gu Howard H
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Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 ,Neurophysiology and neuropsychology ,QP351-495 - Abstract
Abstract Background The dopamine transporter (DAT) plays a critical role in regulating dopamine neurotransmission. Variations in DAT or changes in basal dopaminergic tone have been shown to alter behavior and drug responses. DAT is one of the three known high affinity targets for cocaine, a powerful psychostimulant that produces reward and stimulates locomotor activity in humans and animals. We have shown that cocaine no longer produces reward in knock-in mice with a cocaine insensitive mutant DAT (DAT-CI), suggesting that cocaine inhibition of DAT is critical for its rewarding effect. However, in DAT-CI mice, the mutant DAT has significantly reduced uptake activity resulting in elevated basal dopaminergic tone, which might cause adaptive changes that alter responses to cocaine. Therefore, the objective of this study is to determine how elevated dopaminergic tone affects how mice respond to cocaine. Results We examined the cocaine induced behavior of DAT knockdown mice that have DAT expression reduced by 90% when compared to the wild type mice. Despite a dramatic reduction of DAT expression and marked elevation in basal dopamine tone, cocaine produced reward, as measured by conditioned place preference, and stimulated locomotor activity in these mice. Conclusion A reduction in DAT expression and elevation of dopaminergic tone do not lead to adaptive changes that abolish the rewarding and stimulating effects of cocaine. Therefore, the lack of reward to cocaine observed in DAT-CI mice is unlikely to have resulted from the reduced DAT activity but instead is likely due to the inability of cocaine to block the mutated DAT and increase extracellular dopamine. This study supports the conclusion that the blockade of DAT is required for cocaine reward and locomotor stimulation.
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- 2007
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11. Dilution Enhancement of COS Cell Expression Cloning
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Wu, Xiaohong and Gu, Howard H.
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- 2000
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12. Mechanism for cocaine blocking the transport of dopamine: insights from molecular modeling and dynamics simulations
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Xiaoqin Huang, Gu, Howard H., and Chang-Guo Zhan
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Cocaine -- Chemical properties ,Dopamine -- Chemical properties ,Molecular dynamics -- Usage ,Chemicals, plastics and rubber industries - Published
- 2009
13. Inactivation of the Catalytic Phosphatase Domain of PTPRT/RPTPρ Increases Social Interaction in Mice.
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Thirtamara Rajamani, Keerthi, O'Neill, Brian, Han, Dawn D., Frostholm, Adrienne, Rotter, Andrej, and Gu, Howard H.
- Abstract
Receptor protein tyrosine phosphatase rho ( RPTPρ, gene symbol PTPRT) is a transmembrane protein expressed at high levels in the developing hippocampus, olfactory bulb, cortex, and cerebellum. It has an extracellular domain that interacts with other cell adhesion molecules, and it has two intracellular phosphatase domains, one of which is catalytically active. In a recent genome-wide association study, PTPRT was identified as a potential candidate gene for autism spectrum disorder ( ASD) susceptibility. Mutation of a critical aspartate to alanine ( D1046 A) in the PTPRT catalytic domain inactivates phosphatase function but retains substrate binding. We have generated a knockin mouse line carrying the PTPRT D1046 A mutation. The D1046 A mutation in homozygous knockin mice did not significantly change locomotor activities or anxiety-related behaviors. In contrast, male homozygous mice had significantly higher social approach scores than wild-type animals. Our results suggest that PTPRT phosphatase function is important in modulating neural pathways involved in mouse social behaviors relevant to the symptoms in human ASD patients. Autism Res 2015, 8: 19-28. © 2014 International Society for Autism Research, Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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14. Cause and solutions to the polymerase chain reaction smear problem in genotyping
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Han, Dawn D., Chen, Rong, Hill, Erik R., Tilley, Michael R., and Gu, Howard H.
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- 2006
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15. Effect of serotonin on platelet function in cocaine exposed blood.
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Ziu, Endrit, Hadden, Coedy, Li, Yicong, Lowery III, Curtis Lee, Singh, Preeti, Ucer, Serra S., Mercado, Charles P., Gu, Howard H., and Kilic, Fusun
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SEROTONIN regulation ,SEROTONIN receptors ,BLOOD platelet immunology ,COCAINE ,GENETIC regulation ,PHYSIOLOGY - Abstract
5-hydroxytryptamine (5-HT) reuptake inhibitors counteract the pro-thrombotic effect of elevated plasma 5-HT by down-regulating the 5-HT uptake rates of platelets. Cocaine also down-regulates the platelet 5-HT uptake rates but in contrast, the platelets of cocaine-injected mice show a much higher aggregation rate than the platelets of control mice. To examine the involvement of plasma 5-HT in cocaine-mediated platelet aggregation, we studied the function of platelets isolated from wild-type and transgenic, peripheral 5-HT knock-out (TPH1-KO) mice, and cocaine-insensitive dopamine transporter knock in (DAT-KI) mice. In cocaine-injected mice compared to the control mice, the plasma 5-HT level as well as the surface level of P-selectin was elevated; in vitro platelet aggregation in the presence of type I fibrillar collagen was enhanced. However, cocaine injection lowered the 5-HT uptake rates of platelets and increased the plasma 5-HT levels of the DAT-KI mice but did not change their platelets aggregation rates further which are already hyper-reactive. Furthermore, the in vitro studies supporting these in vivo findings suggest that cocaine mimics the effect of elevated plasma 5-HT level on platelets and in 5-HT receptor- and transporter-dependent pathways in a two-step process propagates platelet aggregation by an additive effect of 5-HT and nonserotonergic catecholamine. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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16. Amphetamine-induced locomotion in a hyperdopaminergic ADHD mouse model depends on genetic background
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O'Neill, Brian and Gu, Howard H.
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TREATMENT of attention-deficit hyperactivity disorder , *DOPAMINE agents , *AMPHETAMINES , *RODENT locomotion , *COCAINE , *LABORATORY mice - Abstract
Abstract: We previously generated a knock-in mouse line with a cocaine-insensitive dopamine transporter (DAT-CI mice). These mice lost several behavioral responses to cocaine, but retained their response to amphetamine. DAT-CI mice are hyperdopaminergic due to reduced DAT function, and may thus be a good model for studying attention deficit hyperactivity disorder (ADHD). These mice had been behaviorally characterized while they were on a mixed genetic background. However as the colony was propagated over time, the mixed genetics were shifted toward a pure C57Bl/6J background — via a common breeding scheme known as “backcrossing.” Several phenotypes appeared to have changed during this time frame. In this study, we investigated whether backcrossing altered the hyperlocomotive phenotype and behavioral responses to amphetamine, a drug used to treat ADHD. C57-congenic DAT-CI mice had high spontaneous locomotor activity that could be suppressed by low doses of amphetamine. Furthermore, their locomotion was not stimulated by very high doses of amphetamine (20mg/kg). After the reversion to a mixed genetic background by breeding with the 129 strain, the C57:129 hybrid DAT-CI mice displayed reduced basal locomotor activity compared to the C57-congenic mutant mice, and regained locomotor stimulation by high-dose amphetamine. The calming effect of amphetamine at low doses was retained in both strains. In summary, reduced DAT function in DAT-CI mice leads to a hyperdopaminergic state, and an ADHD-like phenotype in both strains. The data show that the genetic background of DAT-CI mice affects their locomotor phenotypes and their responses to amphetamine. Since the differences in genetic background between the strains of mice have a significant impact on the ADHD-like phenotype and the response to amphetamine, further study with these strains could identify the genetic underpinnings affecting the severity of ADHD-related symptoms and the treatment response. [Copyright &y& Elsevier]
- Published
- 2013
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17. Specific knockdown of the D2 long dopamine receptor variant.
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Naughton, Bart J., Thirtamara-Rajamani, Keerthi, Wang, Chuansong, During, Matthew J., and Gu, Howard H.
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- 2012
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18. Dopamine Transporter Gene Variant Affecting Expression in Human Brain is Associated with Bipolar Disorder.
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Pinsonneault, Julia K., Han, Dawn D., Burdick, Katherine E., Kataki, Maria, Bertolino, Alessandro, Malhotra, Anil K., Gu, Howard H., and Sadee, Wolfgang
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DOPAMINE ,BIPOLAR disorder ,NEURAL transmission ,COCAINE ,POST-traumatic stress disorder ,CENTRAL nervous system diseases ,MESSENGER RNA - Abstract
The gene encoding the dopamine transporter (DAT) has been implicated in CNS disorders, but the responsible polymorphisms remain uncertain. To search for regulatory polymorphisms, we measured allelic DAT mRNA expression in substantia nigra of human autopsy brain tissues, using two marker SNPs (rs6347 in exon 9 and rs27072 in the 3′-UTR). Allelic mRNA expression imbalance (AEI), an indicator of cis-acting regulatory polymorphisms, was observed in all tissues heterozygous for either of the two marker SNPs. SNP scanning of the DAT locus with AEI ratios as the phenotype, followed by in vitro molecular genetics studies, demonstrated that rs27072 C>T affects mRNA expression and translation. Expression of the minor T allele was dynamically regulated in transfected cell cultures, possibly involving microRNA interactions. Both rs6347 and rs3836790 (intron8 5/6 VNTR) also seemed to affect DAT expression, but not the commonly tested 9/10 VNTR in the 3′UTR (rs28363170). All four polymorphisms (rs6347, intron8 5/6 VNTR, rs27072 and 3′UTR 9/10 VNTR) were genotyped in clinical cohorts, representing schizophrenia, bipolar disorder, depression, and controls. Only rs27072 was significantly associated with bipolar disorder (OR=2.1, p=0.03). This result was replicated in a second bipolar/control population (OR=1.65, p=0.01), supporting a critical role for DAT regulation in bipolar disorder. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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19. Dopamine Transporter Inhibition is Necessary for Cocaine-Induced Increases in Dendritic Spine Density in the Nucleus Accumbens.
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MARTIN, BRADLEY J., NAUGHTON, BARTHOLOMEW J., THIRTAMARA-RAJAMANI, KEERTHI, YOON, DANIEL J., HAN, DAWN D., DEVRIES, A. COURTNEY, and GU, HOWARD H.
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LABORATORY mice ,COCAINE ,NUCLEUS accumbens ,ANIMAL morphology ,SALINE injections - Abstract
Repeated exposure to cocaine produces changes in the nervous system that facilitate drug-seeking behaviors. These drug-seeking behaviors have been studied with animal models, such as cocaine-induced locomotor sensitization. Cocaine is hypothesized to induce locomotor sensitization by neural changes, including an increase in the density of spines on the dendrites of neurons in the nucleus accumbens (NAC). However, how cocaine increases dendritic spine density in the NAC has been difficult to discern because cocaine inhibits the function of multiple targets, including the transporters for dopamine, serotonin, and norepinephrine. Previously, our lab created a tool that is useful for determining how inhibiting the dopamine transporter (DAT) contributes to the effects of cocaine by generating mice that express a cocaine-insensitive DAT (DAT-CI mice). In this study, we used DAT-CI mice to determine the contribution of DAT inhibition in cocaine-induced increases in dendritic spine density in the NAC.We repeatedly injected DAT-CI mice with either cocaine or saline, and measured both dendritic spine density in the NAC and locomotor activity. Unlike wild-type mice, DAT-CI mice did not show an increase in dendritic spine density in the NAC or in locomotor activity inresponse to repeated injections of cocaine. These data show that cocaine-induced increases in dendritic spine density in the NAC require DAT inhibition. Thus, DAT-inhibition may play a role in mediating the long-lasting neural changes associated with drug addiction. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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20. Role of Aberrant Striatal Dopamine D1 Receptor/cAMP/Protein Kinase A/DARPP32 Signaling in the Paradoxical Calming Effect of Amphetamine.
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Napolitano, Francesco, Bonito-Oliva, Alessandra, Federici, Mauro, Carta, Manolo, Errico, Francesco, Magara, Salvatore, Martella, Giuseppina, Nisticò, Robert, Centonze, Diego, Pisani, Antonio, Gu, Howard H., Mercuri, Nicola B., and Usiello, Alessandro
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TREATMENT of attention-deficit hyperactivity disorder ,STIMULANTS ,LABORATORY mice ,DOPAMINE ,PHOSPHORYLATION ,ELECTROPHYSIOLOGY ,THERAPEUTICS - Abstract
Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein, we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP32) at Thr75 residue, but preserved D
2 receptor (D2 R) function. However, although we demonstrated that striatal D1 receptor (D1 R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8-dihydroxy-l-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinase A (PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D1 Rs since haloperidol, by blocking the tonic inhibition of D2 R, unmasked a normal activation of striatal adenosine A2A receptor-mediated cAMP/PKA/DARPP32 cascade in mutants. Most importantly, our studies highlighted that amphetamine, nomifensine, and bupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these drugs in DAT-CI mutants depends on selective aberrant phasic activation of D1 R/cAM P/PKA/ DARPP32 signaling in response to increased striatal extracellular dopamine levels. [ABSTRACT FROM AUTHOR]- Published
- 2010
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21. Potencies of Cocaine Methiodide on Major Cocaine Targets in Mice.
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Hill, Erik R., Tian, Jinbin, Tilley, Michael R., Zhu, Michael X., and Gu, Howard H.
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COCAINE ,NARCOTICS ,DRUG abuse ,TROPANES ,ORGANS (Anatomy) ,NEUROTRANSMITTERS ,TISSUES ,CRYOBIOLOGY ,PHYSIOLOGICAL transport of sodium ,ALKALI metals ,SODIUM channels - Abstract
Cocaine methiodide (CM), a charged cocaine analog, cannot pass the blood brain barrier. It has been assumed the effects of systemic CM represent cocaine actions in peripheral tissues. However, the IC
50 values of CM have not been clearly determined for the major cocaine targets: dopamine, norepinephrine, and serotonin transporters, and sodium channels. Using cells transfected with individual transporters from mice and synaptosomes from mouse striatum tissues, we observed that the inhibition IC50 values for monoamine uptake by CM were 31-fold to 184-fold higher compared to cocaine at each of the transporters. In dorsal root ganglion neurons, cocaine inhibited sodium channels with an apparent IC50 of 75 μM, while CM showed no observable effect at concentrations up to 3 mM. These results indicate that an equal dose of CM will not produce an equivalent peripheral effect of cocaine. [ABSTRACT FROM AUTHOR]- Published
- 2009
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22. Financial and Psychological Risk Attitudes Associated with Two Single Nucleotide Polymorphisms in the Nicotine Receptor (CHRNA4) Gene.
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Roe, Brian E., Tilley, Michael R., Gu, Howard H., Beversdorf, David Q., Sadee, Wolfgang, Haab, Timothy C., and Papp, Audrey C.
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NEUROSCIENCES ,GENETIC polymorphisms ,PSYCHOLOGY ,HETEROGENEITY ,NUCLEOTIDES ,GENOTYPE-environment interaction ,GENES ,SEROTONIN ,DOPAMINE - Abstract
With recent advances in understanding of the neuroscience of risk taking, attention is now turning to genetic factors that may contribute to individual heterogeneity in risk attitudes. In this paper we test for genetic associations with risk attitude measures derived from both the psychology and economics literature. To develop a long-term prospective study, we first evaluate both types of risk attitudes and find that the economic and psychological measures are poorly correlated, suggesting that different genetic factors may underlie human response to risk faced in different behavioral domains. We then examine polymorphisms in a spectrum of candidate genes that affect neurotransmitter systems influencing dopamine regulation or are thought to be associated with risk attitudes or impulsive disorders. Analysis of the genotyping data identified two single nucleotide polymorphisms (SNPs) in the gene encoding the alpha 4 nicotine receptor (CHRNA4, rs4603829 and rs4522666) that are significantly associated with harm avoidance, a risk attitude measurement drawn from the psychology literature. Novelty seeking, another risk attitude measure from the psychology literature, is associated with several COMT (catechol-O-methyl transferase) SNPs while economic risk attitude measures are associated with several VMAT2 (vesicular monoamine transporter) SNPs, but the significance of these associations did not withstand statistical adjustment for multiple testing and requires larger cohorts. These exploratory results provide a starting point for understanding the genetic basis of risk attitudes by considering the range of methods available for measuring risk attitudes and by searching beyond the traditional direct focus on dopamine and serotonin receptor and transporter genes. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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23. Cocaine does not produce reward in absence of dopamine transporter inhibition.
- Author
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Tilley, Michael R., O'neill, Brian, Han, Dawn D., and Gu, Howard H.
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- 2009
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24. Dopamine transporter inhibition is required for cocaine-induced stereotypy.
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Tilley, Michael R. and Gu, Howard H.
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- 2008
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25. Cocaine reward and locomotion stimulation in mice with reduced dopamine transporter expression.
- Author
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Tilley, Michael R., Cagniard, Barbara, Xiaoxi Zhuang, Han, Dawn D., Narry Tiao, and Gu, Howard H.
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COCAINE & psychology ,BRAIN ,LABORATORY mice ,DOPAMINE ,PHARMACOLOGY ,NEUROBIOLOGY - Abstract
Background: The dopamine transporter (DAT) plays a critical role in regulating dopamine neurotransmission. Variations in DAT or changes in basal dopaminergic tone have been shown to alter behavior and drug responses. DAT is one of the three known high affinity targets for cocaine, a powerful psychostimulant that produces reward and stimulates locomotor activity in humans and animals. We have shown that cocaine no longer produces reward in knock-in mice with a cocaine insensitive mutant DAT (DAT-CI), suggesting that cocaine inhibition of DAT is critical for its rewarding effect. However, in DAT-CI mice, the mutant DAT has significantly reduced uptake activity resulting in elevated basal dopaminergic tone, which might cause adaptive changes that alter responses to cocaine. Therefore, the objective of this study is to determine how elevated dopaminergic tone affects how mice respond to cocaine. Results: We examined the cocaine induced behavior of DAT knockdown mice that have DAT expression reduced by 90% when compared to the wild type mice. Despite a dramatic reduction of DAT expression and marked elevation in basal dopamine tone, cocaine produced reward, as measured by conditioned place preference, and stimulated locomotor activity in these mice. Conclusion: A reduction in DAT expression and elevation of dopaminergic tone do not lead to adaptive changes that abolish the rewarding and stimulating effects of cocaine. Therefore, the lack of reward to cocaine observed in DAT-CI mice is unlikely to have resulted from the reduced DAT activity but instead is likely due to the inability of cocaine to block the mutated DAT and increase extracellular dopamine. This study supports the conclusion that the blockade of DAT is required for cocaine reward and locomotor stimulation. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
26. Abolished cocaine reward in mice with a cocaine-insensitive dopamine transporter.
- Author
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Rong Chen, Tilley, Michael R., Hua Wei, Fuwen Zhou, Fu-Ming Zhou, San Ching, Ning Quan, Stephens, Robert L., Hill, Erik R., Nottoli, Timothy, Han, Dawn D., and Gu, Howard H.
- Subjects
COCAINE ,DOPAMINE ,SEROTONIN ,NORADRENALINE ,LOCOMOTION ,MICE - Abstract
There are three known high-affinity targets for cocaine: the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Decades of studies support the dopamine (DA) hypothesis that the blockade of DAT and the subsequent increase in extracellular DA primarily mediate cocaine reward and reinforcement. Contrary to expectations, DAT knockout (DAT-KO) mice and SERT or NET knockout mice still self-administer cocaine and/or display conditioned place preference (CPP) to cocaine, which led to the reevaluation of the DA hypothesis and the proposal of redundant reward pathways. To study the role of DAT in cocaine reward, we have generated a knockin mouse line carrying a functional DAT that is insensitive to cocaine. In these mice, cocaine suppressed locomotor activity, did not elevate extracellular DA in the nucleus accumbens, and did not produce reward as measured by CPP. This result suggests that blockade of DAT is necessary for cocaine reward in mice with a functional DAT. This mouse model is unique in that it is specifically designed to differentiate the role of DAT from the roles of NET and SERT in cocaine-induced biochemical and behavioral effects. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
27. Comparison of the monoamine transporters from human and mouse in their sensitivities to psychostimulant drugs.
- Author
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Han, Dawn D. and Gu, Howard H.
- Subjects
NEUROTRANSMITTERS ,NEURAL transmission ,PSYCHIATRIC drugs ,DRUGS of abuse ,AMPHETAMINES ,PHARMACODYNAMICS - Abstract
Background: The plasma membrane neurotransmitter transporters terminate neurotransmissions by the reuptake of the released neurotransmitters. The transporters for the monoamines dopamine, norepinephrine, and serotonin (DAT, NET, and SERT) are targets for several popular psychostimulant drugs of abuse. The potencies of the psychostimulant on the monoamine transporters have been studied by several laboratories. However, there are significant discrepancies in the reported data with differences up to 60-fold. In addition, the drug potencies of the 3 monoamine transporters from mouse have not been compared in the same experiments or along side the human transporters. Further studies and systematic comparisons are needed. Results: In this study, we compared the potencies of five psychostimulant drugs to inhibit human and mouse DAT, SERT and NET in the same cellular background. The K
I values of cocaine to inhibit the 3 transporters are within a narrow range of 0.2 to 0.7 μM. In comparison, methylphenidate inhibited DAT and NET at around 0.1 μM, while it inhibited SERT at around 100 μM. The order of amphetamine potencies was NET (KI = 0.07-0.1 μM), DAT (KI = 0.6 μM), and SERT (KI between 20 to 40 μM). The results for methamphetamine were similar to those for amphetamine. In contrast, another amphetamine derivative, MDMA (3-4 methylenedioxymethamphetamine), exhibited higher potency at SERT than at DAT. The human and mouse transporters were similar in their sensitivities to each of the tested drugs (KI values are within 4-fold). Conclusion: The current and previous studies support the following conclusions: 1) cocaine blocks all 3 monoamine transporters at similar concentrations; 2 methylphenidate inhibits DAT and NET well but a 1000-fold higher concentration of the drug is required to inhibit SERT; 3) Amphetamine and methamphetamine are most potent at NET, while being 5- to 9-fold less potent at DAT, and 200- to 500-fold less potent at SERT; 4) MDMA has moderately higher apparent affinity for SERT and NET than for DAT. The relative potencies of a drug to inhibit DAT, NET and SERT suggest which neurotransmitter systems are disrupted the most by each of these stimulants and thus the likely primary mechanism of drug action. [ABSTRACT FROM AUTHOR]- Published
- 2006
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28. A triple mutation in the second transmembrane domain of mouse dopamine transporter markedly decreases sensitivity to cocaine and methylphenidate.
- Author
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Rong Chen, Han, Dawn D., and Gu, Howard H.
- Subjects
DOPAMINE ,NEUROTRANSMITTERS ,COCAINE ,CATECHOLAMINES ,AMPHETAMINES ,BIOCHEMISTRY - Abstract
Previously, we reported that Phe105 in transmembrane domain 2 of the mouse dopamine transporter (DAT) is crucial for high-affinity cocaine binding. In the current study, we investigated whether other residues surrounding Phe105 also affect the potency of cocaine inhibition. After three rounds of sequential random mutagenesis at these residues, we found a triple mutant (L104V, F105C and A109V) of mouse DAT that retained over 50% uptake activity and was 69-fold less sensitive to cocaine inhibition when compared with the wild-type mouse DAT. The triple mutation also resulted in a 47-fold decrease in sensitivity to methylphenidate inhibition, suggesting that the binding sites for cocaine and methylphenidate may overlap. In contrast, the inhibition of dopamine uptake by amphetamine or methamphetamine was not significantly changed by the mutations, suggesting that the binding sites for the amphetamines differ from those for cocaine and methylphenidate. Such functional but cocaine-insensitive DAT mutants can be used to generate a knock-in mouse line to study the role of DAT in cocaine addiction. [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
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29. Functional mutations in mouse norepinephrine transporter reduce sensitivity to cocaine inhibition
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Wei, Hua, Hill, Erik R., and Gu, Howard H.
- Subjects
- *
MUTAGENESIS , *NORADRENALINE , *DRUG carriers , *DRUG resistance , *COCAINE , *LABORATORY mice , *ANTIDEPRESSANTS , *THERAPEUTICS - Abstract
Abstract: The transporters of dopamine, norepinephrine and serotonin are molecular targets of cocaine, amphetamine, and therapeutic antidepressants. The residues involved in binding these drugs are unknown. We have performed several rounds of random and site-directed mutagenesis in the mouse norepinephrine transporter and screened for mutants with altered sensitivity to cocaine inhibition of substrate uptake. We have identified a triple mutation that retains close to wild-type transport function but displays a 37-fold decrease in cocaine sensitivity and 24-fold decrease in desipramine sensitivity. In contrast, the mutant''s sensitivities to amphetamine, methamphetamine, and methylphenidate are only slightly changed. Our data reveal critical residues contributing to the potent uptake inhibitions by these important drugs. Furthermore, this drug-resistant triple mutant can be used to generate a unique knock-in mouse line to study the role of norepinephrine transporter in the addictive effects of cocaine and the therapeutic effects of desipramine. [Copyright &y& Elsevier]
- Published
- 2009
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30. Fluorescence-based evaluation of shRNA efficacy
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Naughton, B.J., Han, Dawn D., and Gu, Howard H.
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FLUORESCENCE , *NON-coding RNA , *MESSENGER RNA , *SMALL interfering RNA , *MEDICAL screening , *GENE silencing , *GENE targeting , *NUCLEOTIDE sequence , *CELLULAR control mechanisms - Abstract
Abstract: RNA interference is a cellular mechanism regulating levels of mRNAs. It has been widely exploited to knock down specific protein targets. The selected interfering RNA sequence greatly influences its ability to knock down the target. Here we present a method for constructing multiple testing plasmids which express small hairpin RNAs (shRNA) targeting different regions of an mRNA. A simple fluorescence test in cultured cells allows convenient evaluation of mRNA knockdown by many different shRNAs on 96-well plates. We show that software predicted shRNAs have varying efficacies and only 2 of the 7 tested shRNAs significantly knocked down their targets. [Copyright &y& Elsevier]
- Published
- 2011
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- View/download PDF
31. Behavior of knock-in mice with a cocaine-insensitive dopamine transporter after virogenetic restoration of cocaine sensitivity in the striatum.
- Author
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O'Neill, Brian, Tilley, Michael R., Han, Dawn D., Thirtamara-Rajamani, Keerthi, Hill, Erik R., Bishop, Georgia A., Zhou, Fu-Ming, During, Matthew J., and Gu, Howard H.
- Subjects
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COCAINE , *DOPAMINE , *NORADRENALINE , *SEROTONIN transporters , *DRUG addiction , *LABORATORY mice , *GENE expression , *PHYSIOLOGY - Abstract
Abstract: Cocaine's main pharmacological actions are the inhibition of the dopamine, serotonin, and norepinephrine transporters. Its main behavioral effects are reward and locomotor stimulation, potentially leading to addiction. Using knock-in mice with a cocaine-insensitive dopamine transporter (DAT-CI mice) we have shown previously that inhibition of the dopamine transporter (DAT) is necessary for both of these behaviors. In this study, we sought to determine brain regions in which DAT inhibition by cocaine stimulates locomotor activity and/or produces reward. We used adeno-associated viral vectors to re-introduce the cocaine-sensitive wild-type DAT in specific brain regions of DAT-CI mice, which otherwise only express a cocaine-insensitive DAT globally. Viral-mediated expression of wild-type DAT in the rostrolateral striatum restored cocaine-induced locomotor stimulation and sensitization in DAT-CI mice. In contrast, the expression of wild-type DAT in the dorsal striatum, or in the medial nucleus accumbens, did not restore cocaine-induced locomotor stimulation. These data help to determine cocaine's molecular actions and anatomical loci that cause hyperlocomotion. Interestingly, cocaine did not produce significant reward – as measured by conditioned place-preference – in any of the three cohorts of DAT-CI mice with the virus injections. Therefore, the locus or loci underlying cocaine-induced reward remain underdetermined. It is possible that multiple dopamine-related brain regions are involved in producing the robust rewarding effect of cocaine. [Copyright &y& Elsevier]
- Published
- 2014
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32. Interaction of tyrosine 151 in norepinephrine transporter with the 2β group of cocaine analog RTI-113
- Author
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Hill, Erik R., Huang, Xiaoqin, Zhan, Chang-Guo, Ivy Carroll, F., and Gu, Howard H.
- Subjects
- *
TYROSINE , *NORADRENERGIC mechanisms , *COCAINE , *ELECTROSTATICS , *SEROTONIN , *DOPAMINE , *BINDING sites , *COMPUTER simulation - Abstract
Abstract: Cocaine binds and inhibits dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter. The residues forming cocaine binding sites are unknown. RTI-113, a cocaine analog, is 100× more potent at inhibiting DAT than inhibiting NET. Here we show that removing the hydroxyl group from residue Tyr151 in NET by replacing it with Phe, the corresponding residue in DAT, increased the sensitivity of NET to RTI-113, while the reverse mutation in DAT decreased the sensitivity of DAT to RTI-113. In contrast, RTI-31, another cocaine analog having the same structure as RTI-113 but with the phenyl group at the 2β position replaced by a methyl group, inhibits the transporter mutants equally well whether a hydroxyl group is present at the residue or not. The data suggest that this residue contributes to cocaine binding site and is close to the 2β position of cocaine analogs. These results are consistent with our previously proposed cocaine-DAT binding model where cocaine initially binds to a site that does not overlap with, but is close to, the dopamine-binding site. Computational modeling and molecular docking yielded a binding model that explains the observed changes in RTI-113 inhibition potencies. [Copyright &y& Elsevier]
- Published
- 2011
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33. Molecular cloning and functional characterization of the dopamine transporter from Eloria noyesi, a caterpillar pest of cocaine-rich coca plants
- Author
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Chen, Rong, Wu, Xiaohong, Wei, Hua, Han, Dawn D., and Gu, Howard H.
- Subjects
- *
COCAINE , *COCA , *INSECTS , *INSECT-plant relationships , *DOPAMINE - Abstract
Abstract: Cocaine is produced by coca plants as a chemical defense to deter feeding by insects. It has been shown that cocaine sprayed on tomato leaves reduces insect feeding, causes abnormal behaviors at low doses and kills feeding insects at doses equivalent to that in coca leaves [Nathanson, J.A., Hunnicutt, E.J., Kantham, L., Scavone, C., 1993. Cocaine as a naturally occurring insecticide. Proc. Natl. Acad. Sci. U. S. A. 90, 9645–9648.]. Most insects avoid coca leaves except the larvae of Eloria noyesi, a caterpillar pest of coca plants, which feeds preferentially on coca leaves. In the current study, we cloned and characterized the dopamine transporters (DATs) from caterpillars of E. noyesi (enDAT) and the silkworm, Bombyx mori (B. mori, bmDAT). The two insect DATs shared 88% amino acid sequence homology and functional similarity. Although enDAT and bmDAT showed the highest affinity for dopamine among endogenous amines, they were more sensitive to mammalian NET-selective inhibitors than to mammalian DAT-selective inhibitors. Despite a high cocaine content in the food source for E. noyesi, cocaine sensitivity of enDAT was similar to that of bmDAT, suggesting that mechanisms other than DAT insensitivity to cocaine, such as cocaine sequestration, might be responsible for cocaine resistance in this species. Given the significant differences in pharmacological profile from mammalian DATs, invertebrate DATs provide excellent tools for identifying regions and residues in the transporters that contribute to high-affinity binding of psychostimulants and antidepressants. [Copyright &y& Elsevier]
- Published
- 2006
- Full Text
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34. Inactivation of the catalytic phosphatase domain of PTPRT/RPTPρ increases social interaction in mice.
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Thirtamara Rajamani K, O'Neill B, Han DD, Frostholm A, Rotter A, and Gu HH
- Subjects
- Analysis of Variance, Animals, Female, Male, Mice, Mutation, Sex Factors, Behavior, Animal physiology, Receptor-Like Protein Tyrosine Phosphatases, Class 2 genetics
- Abstract
Receptor protein tyrosine phosphatase rho (RPTPρ, gene symbol PTPRT) is a transmembrane protein expressed at high levels in the developing hippocampus, olfactory bulb, cortex, and cerebellum. It has an extracellular domain that interacts with other cell adhesion molecules, and it has two intracellular phosphatase domains, one of which is catalytically active. In a recent genome-wide association study, PTPRT was identified as a potential candidate gene for autism spectrum disorder (ASD) susceptibility. Mutation of a critical aspartate to alanine (D1046A) in the PTPRT catalytic domain inactivates phosphatase function but retains substrate binding. We have generated a knockin mouse line carrying the PTPRT D1046A mutation. The D1046A mutation in homozygous knockin mice did not significantly change locomotor activities or anxiety-related behaviors. In contrast, male homozygous mice had significantly higher social approach scores than wild-type animals. Our results suggest that PTPRT phosphatase function is important in modulating neural pathways involved in mouse social behaviors relevant to the symptoms in human ASD patients., (© 2014 International Society for Autism Research, Wiley Periodicals, Inc.)
- Published
- 2015
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35. Role of aberrant striatal dopamine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine.
- Author
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Napolitano F, Bonito-Oliva A, Federici M, Carta M, Errico F, Magara S, Martella G, Nisticò R, Centonze D, Pisani A, Gu HH, Mercuri NB, and Usiello A
- Subjects
- Animals, Corpus Striatum physiology, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Discrimination, Psychological drug effects, Discrimination, Psychological physiology, Dopamine metabolism, Dopamine Plasma Membrane Transport Proteins genetics, Dopamine and cAMP-Regulated Phosphoprotein 32 metabolism, Gene Knock-In Techniques, Long-Term Potentiation drug effects, Long-Term Potentiation physiology, Male, Mice, Mice, Transgenic, Motor Activity physiology, Mutation, Random Allocation, Receptors, Dopamine D1 metabolism, Receptors, Dopamine D2 metabolism, Amphetamine pharmacology, Central Nervous System Stimulants pharmacology, Corpus Striatum drug effects, Motor Activity drug effects, Signal Transduction
- Abstract
Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein, we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP32) at Thr75 residue, but preserved D(2) receptor (D(2)R) function. However, although we demonstrated that striatal D(1) receptor (D(1)R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinase A (PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D(1)Rs since haloperidol, by blocking the tonic inhibition of D(2)R, unmasked a normal activation of striatal adenosine A(2A) receptor-mediated cAMP/PKA/DARPP32 cascade in mutants. Most importantly, our studies highlighted that amphetamine, nomifensine, and bupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these drugs in DAT-CI mutants depends on selective aberrant phasic activation of D(1)R/cAMP/PKA/DARPP32 signaling in response to increased striatal extracellular dopamine levels.
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- 2010
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36. Mechanism for cocaine blocking the transport of dopamine: insights from molecular modeling and dynamics simulations.
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Huang X, Gu HH, and Zhan CG
- Subjects
- Binding Sites, Biological Transport, Dopamine Plasma Membrane Transport Proteins antagonists & inhibitors, Kinetics, Models, Molecular, Cocaine pharmacology, Dopamine metabolism, Molecular Dynamics Simulation
- Abstract
Molecular modeling and dynamics simulations have been performed to study how cocaine inhibits dopamine transporter (DAT) for the transport of dopamine. The computationally determined DAT-ligand binding mode is totally different from the previously proposed overlap binding mode in which cocaine- and dopamine-binding sites are the same (Beuming, T.; et al. Nat. Neurosci. 2008, 11, 780-789). The new cocaine-binding site does not overlap with, but is close to, the dopamine-binding site. Analysis of all results reveals that when cocaine binds to DAT, the initial binding site is likely the one modeled in this study because this binding site can naturally accommodate cocaine. Then cocaine may move to the dopamine-binding site after DAT makes some necessary conformational change and expands the binding site cavity. It has been demonstrated that cocaine may inhibit the transport of dopamine through both blocking the initial DAT-dopamine binding and reducing the kinetic turnover of the transporter following the DAT-dopamine binding. The relative contributions to the phenomenological inhibition of the transport of dopamine from blocking the initial binding and reducing the kinetic turnover can be different in different types of assays. The obtained general structural and mechanistic insights are consistent with available experimental data and could be valuable for guiding future studies toward understanding cocaine's inhibiting of other transporters.
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- 2009
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37. Lack of cocaine self-administration in mice expressing a cocaine-insensitive dopamine transporter.
- Author
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Thomsen M, Han DD, Gu HH, and Caine SB
- Subjects
- Animals, Cocaine-Related Disorders genetics, Cocaine-Related Disorders prevention & control, Dopamine Plasma Membrane Transport Proteins genetics, Female, Gene Expression, Gene Knock-In Techniques, Male, Mice, Mice, Mutant Strains, Random Allocation, Reinforcement Schedule, Self Administration, Cocaine administration & dosage, Cocaine-Related Disorders metabolism, Dopamine Plasma Membrane Transport Proteins biosynthesis
- Abstract
Cocaine addiction is a worldwide public health problem for which there are no established treatments. The dopamine transporter (DAT) is suspected as the primary target mediating cocaine's abuse-related effects based on numerous pharmacological studies. However, in a previous study, DAT knockout mice were reported to self-administer cocaine, generating much debate regarding the importance of the DAT in cocaine's abuse-related effects. Here, we show that mice expressing a "knockin" of a cocaine-insensitive but functional DAT did not self-administer cocaine intravenously despite normal food-maintained responding and normal intravenous self-administration of amphetamine and a direct dopamine agonist. Our results have three implications. First, they imply a crucial role for high-affinity DAT binding of cocaine in mediating its reinforcing effects, reconciling mouse genetic engineering approaches with data from classic pharmacological studies. Second, they demonstrate the usefulness of knockin strategies that modify specific amino acid sequences within a protein. Third, they show that it is possible to alter the DAT protein sequence in such a way as to selectively target its interaction with cocaine, while sparing other behaviors dependent on DAT function. Thus, molecular engineering technology could advance the development of highly specialized compounds such as a dopamine-sparing "cocaine antagonist."
- Published
- 2009
- Full Text
- View/download PDF
38. The effects of methylphenidate on knockin mice with a methylphenidate-resistant dopamine transporter.
- Author
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Tilley MR and Gu HH
- Subjects
- Animals, Cocaine pharmacology, Conditioning, Psychological drug effects, Dopamine metabolism, Dopamine Plasma Membrane Transport Proteins genetics, Drug Resistance, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Motor Activity drug effects, Norepinephrine Plasma Membrane Transport Proteins antagonists & inhibitors, Stereotyped Behavior drug effects, Dopamine Plasma Membrane Transport Proteins antagonists & inhibitors, Methylphenidate pharmacology
- Abstract
Methylphenidate (Ritalin) is one of the most commonly abused prescription drugs. It is a psychostimulant that inhibits the dopamine and norepinephrine transporters with high affinity. In mice, methylphenidate stimulates locomotor activity, is self-administered, and produces conditioned place preference, typical properties of an addictive drug. We have generated a knockin mouse line bearing a mutant dopamine transporter that is approximately 80-fold less sensitive to cocaine inhibition than wild type. It is interesting to note that this mutant is also almost 50-fold less sensitive to methylphenidate inhibition, suggesting similarities in the binding site for cocaine and methylphenidate. Because methylphenidate is not effective at inhibiting the mutant dopamine transporter, we hypothesized that it would not stimulate locomotor activity or produce reward in the knockin mice. In these knockin mice, doses up to 40 mg/kg methylphenidate either inhibit or fail to stimulate locomotor activity and do not produce conditioned place preference. Doses up to 40 mg/kg methylphenidate also fail to produce stereotypy in the knockin mice. Nisoxetine and desipramine, selective norepinephrine transporter inhibitors, also reduce locomotor activity in wild-type and knockin mice. These results indicate that enhanced dopaminergic neurotransmission is required for methylphenidate's stimulating and rewarding effects. In addition, we observed that drugs enhancing noradrenergic neurotransmission inhibit locomotor activity in mice, which is consistent with the notion that methylphenidate's ability to inhibit the norepinephrine transporter may contribute to its efficacy in treating attention deficit hyperactivity disorder.
- Published
- 2008
- Full Text
- View/download PDF
39. Conserved serine residues in serotonin transporter contribute to high-affinity cocaine binding.
- Author
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Gu HH, Wu X, and Han DD
- Subjects
- Amino Acid Sequence, Animals, Conserved Sequence, Drosophila Proteins genetics, Drosophila Proteins metabolism, HeLa Cells, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Serine genetics, Serotonin Plasma Membrane Transport Proteins genetics, Threonine genetics, Threonine metabolism, Bombyx metabolism, Cocaine metabolism, Serine metabolism, Serotonin Plasma Membrane Transport Proteins metabolism
- Abstract
Serotonin transporter (SERT) is one of the key protein targets of cocaine. Despite intensive studies, it is not clear where cocaine binds to its targets and what residues are involved in cocaine binding. We have cloned the serotonin transporter from silkworm (Bombyx mori, bmSERT). When expressed in cultured cells, bmSERT is over 20-fold less sensitive to cocaine than Drosophila melanogaster SERT (dmSERT). We performed species-scanning mutagenesis using bmSERT and dmSERT. There are two adjacent threonine residues in transmembrane domain 12 of bmSERT where the corresponding residues are two serines in dmSERT and in all known mammalian monoamine transporters. Replacing the serine residues with threonines in dmSERT reduces cocaine sensitivity; while switching the two threonine residues in bmSERT to serines increased cocaine sensitivity. Mutations at the corresponding residues in dopamine transporter also changed cocaine affinity. Our results suggest that the conserved serine residues in SERT contribute to high-affinity cocaine binding.
- Published
- 2006
- Full Text
- View/download PDF
40. Cocaine affinity decreased by mutations of aromatic residue phenylalanine 105 in the transmembrane domain 2 of dopamine transporter.
- Author
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Wu X and Gu HH
- Subjects
- Animals, DNA, Complementary analysis, Dopamine Plasma Membrane Transport Proteins, Drosophila melanogaster, Membrane Proteins drug effects, Membrane Proteins genetics, Membrane Proteins metabolism, Membrane Transport Proteins drug effects, Membrane Transport Proteins genetics, Mice, Phenylalanine genetics, Point Mutation, Protein Structure, Tertiary, Recombinant Fusion Proteins drug effects, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Cocaine pharmacology, Membrane Glycoproteins, Membrane Transport Proteins metabolism, Nerve Tissue Proteins, Phenylalanine metabolism
- Abstract
Dopamine transporter (DAT) is a major target of cocaine, one of the most abused drugs. Major efforts have been focused on defining residues in DAT involved in cocaine binding. We have isolated the Drosophila melanogaster DAT (dDAT) cDNA, which is 10-fold less sensitive to cocaine than the mammalian DATs. Replacing transmembrane domain 2 (TM2) of mouse DAT (mDAT) with dDAT sequence reduced cocaine sensitivity. The reciprocal construct exhibited increased cocaine sensitivity. Switching residue 105 in TM2, a phenylalanine conserved in all mammalian DATs, to methionine, the corresponding residue in dDAT, resulted in a functional transporter with cocaine sensitivity 4-fold lower. Replacing F105 with alanine, leucine, isoleucine, serine, threonine, asparagine, or glutamine resulted in transporters with low transport activity. In contrast, changing F105 to the other aromatic residues tyrosine or tryptophan retained more than 75% transport activity and high cocaine sensitivity. Most significantly, the reciprocal construct, switching the methionine in dDAT at the corresponding residue to phenylalanine, increased cocaine sensitivity 3-fold. Finally, the mDAT mutant with a cysteine at this position had normal transport activity but exhibited cocaine sensitivity that was 15-fold lower. These results suggest that F105 in mDAT contributes to high-affinity cocaine binding. The functional cocaine-insensitive mutants provide tools for the study of the mechanism of cocaine addiction.
- Published
- 2003
- Full Text
- View/download PDF
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