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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.

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]

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]

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 D2 receptor (D2R) function. However, although we demonstrated that striatal D1 receptor (D1R) 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 D1Rs since haloperidol, by blocking the tonic inhibition of D2R, 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 D1R/cAM P/PKA/ DARPP32 signaling in response to increased striatal extracellular dopamine levels. [ABSTRACT FROM AUTHOR]

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]

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]

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]

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.)

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.