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The nematode Caenorhabditis elegans is well known for its ability to support forward genetic screens to identify molecules involved in neuronal viability and signaling. The proteins involved in C. elegans dopamine (DA) regulation are highly conserved across evolution, with prior work demonstrating that the model can serve as an efficient platform to identify novel genes involved in disease-associated processes. To identify novel players in DA signaling, we took advantage of a recently developed library of pre-sequenced mutant nematodes arising from the million mutation project (MMP) to identify strains that display the DA-dependent swimming-induced-paralysis phenotype (Swip). Our screen identified novel mutations in the dopamine transporter encoding gene dat-1, whose loss was previously used to identify the Swip phenotype, as well as multiple genes with previously unknown connections to DA signaling. Here, we present our isolation and characterization of one of these genes, bbs-1, previously linked to the function of primary cilia in worms and higher organisms, including humans, and where loss-of-function mutations result in a human disorder known as Bardet-Biedl syndrome. Our studies of C. elegans BBS-1 protein, as well as other proteins that are known to be assembled into a higher order complex (the BBSome) reveal that functional or structural disruption of this complex leads to exaggerated C. elegans DA signaling to produce Swip via a cell-autonomous mechanism. We provide evidence that not only does the proper function of cilia in C. elegans DA neurons support normal swimming behavior, but also that bbs-1 maintains normal levels of DAT-1 trafficking or function via a RHO-1 and SWIP-13/MAPK-15 dependent pathway where mutants may contribute to Swip independent of altered ciliary function. Together, these studies demonstrate novel contributors to DA neuron function in the worm and demonstrate the utility and efficiency of forward genetic screens using the MMP library., (© 2024 International Society for Neurochemistry.)

Increasing extracellular levels of serotonin (5-HT) in the brain ameliorates symptoms of depression and anxiety-related disorders, e.g., social phobias and post-traumatic stress disorder. Recent evidence from preclinical and clinical studies established the therapeutic potential of drugs inducing the release of 5-HT via the 5-HT-transporter. Nevertheless, current 5-HT releasing compounds under clinical investigation carry the risk for abuse and deleterious side effects. Here, we demonstrate that S-enantiomers of certain ring-substituted cathinones show preference for the release of 5-HT ex vivo and in vivo, and exert 5-HT-associated effects in preclinical behavioral models. Importantly, the lead cathinone compounds (1) do not induce substantial dopamine release and (2) display reduced off-target activity at vesicular monoamine transporters and 5-HT 2B -receptors, indicative of low abuse-liability and low potential for adverse events. Taken together, our findings identify these agents as lead compounds that may prove useful for the treatment of disorders where elevation of 5-HT has proven beneficial., (© 2022. The Author(s).)

Virtually all neuropsychiatric disorders display sex differences in prevalence, age of onset, and/or clinical symptomology. Although altered dopamine (DA) signaling is a feature of many of these disorders, sex-dependent mechanisms uniquely responsive to DA that drive sex-dependent behaviors remain unelucidated. Previously, we established that anomalous DA efflux (ADE) is a prominent feature of the DA transporter (DAT) variant Val559, a coding substitution identified in two male-biased disorders: attention-deficit/hyperactivity disorder and autism spectrum disorder. In vivo, Val559 ADE induces activation of nigrostriatal D2-type DA autoreceptors (D2ARs) that magnifies inappropriate, nonvesicular DA release by elevating phosphorylation and surface trafficking of ADE-prone DAT proteins. Here we demonstrate that DAT Val559 mice exhibit sex-dependent alterations in psychostimulant responses, social behavior, and cognitive performance. In a search for underlying mechanisms, we discovered that the ability of ADE to elicit D2AR regulation of DAT is both sex and circuit-dependent, with dorsal striatum D2AR/DAT coupling evident only in males, whereas D2AR/DAT coupling in the ventral striatum is exclusive to females. Moreover, systemic administration of the D2R antagonist sulpiride, which precludes ADE-driven DAT trafficking, can normalize DAT Val559 behavioral changes unique to each sex and without effects on the opposite sex or wildtype mice. Our studies support the sex- and circuit dependent capacity of D2ARs to regulate DAT as a critical determinant of the sex-biased effects of perturbed DA signaling in neurobehavioral disorders. Moreover, our work provides a cogent example of how a shared biological insult drives alternative physiological and behavioral trajectories as opposed to resilience., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Various stimuli have been employed as reinforcers in preclinical rodent models to elucidate the underpinnings of reward at a molecular and circuit level, with the release of dopamine (DA) in the nucleus accumbens (NAc) as a well-replicated, physiological correlate. Many factors, however, including strain differences, sex, prior stress, and reinforcer administration protocols can influence reward responding and DA release. Although previous evidence indicates that access to the home cage can be an effective reinforcer in behavioral tasks, whether this simple environmental manipulation can trigger DA release in the NAc has not been demonstrated. Here, using fiber photometric recordings of in vivo NAc dopamine release from a genetically-encoded DA sensor, we show that the movement of animals from the home cage to a clear, polycarbonate recording chamber evokes little to no DA release following initial exposure whereas returning animals from the recording chamber to a clean, home-like cage or to the home cage robustly triggers the release of DA, comparable in size to that observed with a 10 mg/kg i.p. Cocaine injection in the recording chamber. Although DA release can be evoked in moving mice to a clean cage, this release was significantly augmented when moving animals from the clean cage to the home cage. Our data provide direct evidence that home cage return from a foreign environment results in a biochemical change consistent with that of a rewarding stimulus. This simple environmental manipulation provides a minimally invasive approach to study the reward circuitry underlying an ethologically relevant reinforcer, return to the safe confines of "home". The home cage - DA release paradigm may also represent a biomarker-driven paradigm for the evaluation of genetic and experiential events that underlie anhedonic states, characteristic of major mood disorders, and to present new opportunities to identify their treatments., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dopamine (DA) signaling is critical to the modulation of multiple brain functions including locomotion, reinforcement, attention and cognition. The literature provides strong evidence that altered DA availability and actions can impact normal neurodevelopment, with both early and enduring consequences on anatomy, physiology and behavior. An appreciation for the developmental contributions of DA signaling to brain development is needed to guide efforts to preclude and remedy neurobehavioral disorders, such as attention-deficit/hyperactivity disorder, addiction, bipolar disorder, schizophrenia and autism spectrum disorder, each of which exhibits links to DA via genetic, cellular and/or pharmacological findings. In this review, we highlight research pursued in preclinical models that use genetic and pharmacological approaches to manipulate DA signaling at sensitive developmental stages, leading to changes at molecular, circuit and/or behavioral levels. We discuss how these alterations can be aligned with traits displayed by neuropsychiatric diseases. Lastly, we review human studies that evaluate contributions of developmental perturbations of DA systems to increased risk for neuropsychiatric disorders., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dopamine (DA) is a neurotransmitter with actions across phylogeny that modulate core behaviors such as motor activity, reward, attention, and cognition. Perturbed DA signaling in humans is associated with multiple disorders, including addiction, ADHD, schizophrenia, and Parkinson's disease. The presynaptic DA transporter exerts powerful control on DA signaling by efficient clearance of the neurotransmitter following release. As in vertebrates, Caenorhabditis elegans DAT (DAT-1) constrains DA signaling and loss of function mutations in the dat-1 gene result in slowed crawling on solid media and swimming-induced paralysis (Swip) in water. Previously, we identified a mutant line, vt34, that exhibits robust DA-dependent Swip. vt34 exhibits biochemical and behavioral phenotypes consistent with reduced DAT-1 function though vt34; dat-1 double mutants exhibit an enhanced Swip phenotype, suggesting contributions of the vt34-associated mutation to additional mechanisms that lead to excess DA signaling. SNP mapping and whole genome sequencing of vt34 identified the site of the molecular lesion in the gene B0412.2 that encodes the Runx transcription factor ortholog RNT-1. Unlike dat-1 animals, but similar to other loss of function rnt-1 mutants, vt34 exhibits altered male tail morphology and reduced body size. Deletion mutations in both rnt-1 and the bro-1 gene, which encodes a RNT-1 binding partner also exhibit Swip. Both vt34 and rnt-1 mutations exhibit reduced levels of dat-1 mRNA as well as the tyrosine hydroxylase ortholog cat-2. Although reporter studies indicate that rnt-1 is expressed in DA neurons, its re-expression in DA neurons of vt34 animals fails to fully rescue Swip. Moreover, as shown for vt34, rnt-1 mutation exhibits additivity with dat-1 in generating Swip, as do rnt-1 and bro-1 mutations, and vt34 exhibits altered capacity for acetylcholine signaling at the neuromuscular junction. Together, these findings identify a novel role for rnt-1 in limiting DA neurotransmission and suggest that loss of RNT-1 may disrupt function of both DA neurons and body wall muscle to drive Swip., Competing Interests: The authors have declared that no competing interests exist.

Dopamine (DA) signaling dysfunction is believed to contribute to multiple neuropsychiatric disorders including attention-deficit/hyperactivity disorder (ADHD). The rare DA transporter (DAT) coding substitution Ala559Val found in subjects with ADHD, bipolar disorder and autism, promotes anomalous DA efflux in vitro and, in DAT Val559 mice, leads to increased reactivity to imminent handling, waiting impulsivity, and enhanced motivation for reward. Here, we report that, in contrast to amphetamine and methylphenidate, which induce significant locomotor activation, cocaine administration to these mice elicits no locomotor effects, despite retention of conditioned place preference (CPP). Additionally, cocaine fails to elevate extracellular DA. Given that amphetamine and methylphenidate, unlike cocaine, lack high-affinity interactions with the serotonin (5-HT) transporter (SERT), we hypothesized that the lack of cocaine-induced hyperlocomotion in DAT Val559 mice arises from SERT blockade and augmented 5-HT signaling relative to cocaine actions on wildtype animals. Consistent with this idea, the SERT blocker fluoxetine abolished methylphenidate-induced locomotor activity in DAT Val559 mice, mimicking the effects seen with cocaine. Additionally, a cocaine analog (RTI-113) with greater selectivity for DAT over SERT retains locomotor activation in DAT Val559 mice. Furthermore, genetic elimination of high-affinity cocaine interactions at SERT in DAT Val559 mice, or specific inhibition of 5-HT 2C receptors in these animals, restored cocaine-induced locomotion, but did not restore cocaine-induced elevations of extracellular DA. Our findings reveal a significant serotonergic plasticity arising in the DAT Val559 model that involves enhanced 5-HT 2C signaling, acting independently of striatal DA release, capable of suppressing the activity of cocaine-sensitive motor circuits.

Disruptions of dopamine (DA) signaling contribute to a broad spectrum of neuropsychiatric disorders, including attention-deficit hyperactivity disorder (ADHD), addiction, bipolar disorder, and schizophrenia. Despite evidence that risk for these disorders derives from heritable variation in DA-linked genes, a better understanding is needed of the molecular and circuit context through which gene variation drives distinct disease traits. Previously, we identified the DA transporter (DAT) variant Val559 in subjects with ADHD and established that the mutation supports anomalous DAT-mediated DA efflux (ADE). Here, we demonstrate that region-specific contributions of D 2 autoreceptors (D2AR) to presynaptic DA homeostasis dictate the consequences of Val559 expression in adolescent male mice. We show that activation of D2ARs in the WT dorsal striatum (DS), but not ventral striatum (VS), increases DAT phosphorylation and surface trafficking. In contrast, the activity of tyrosine hydroxylase (TH) is D2AR-dependent in both regions. In the DS but not VS of Val559 mice, tonic activation of D2ARs drives a positive feedback loop that promotes surface expression of efflux-prone DATs, raising extracellular DA levels and overwhelming DAT-mediated DA clearance capacity. Whereas D2ARs that regulate DAT are tonically activated in the Val559 DS, D2ARs that regulate TH become desensitized, allowing maintenance of cytosolic DA needed to sustain ADE. Together with prior findings, our results argue for distinct D2AR pools that regulate DA synthesis versus DA release and inactivation and offer a clear example of how the penetrance of gene variation can be limited to a subset of expression sites based on differences in intersecting regulatory networks. SIGNIFICANCE STATEMENT Altered dopamine (DA) signaling has been linked to multiple neuropsychiatric disorders. In an effort to understand and model disease-associated DAergic disturbances, we previously screened the DA transporter (DAT) in subjects with attention-deficit hyperactivity disorder (ADHD) and identified multiple, functionally impactful, coding variants. One of these variants, Val559, supports anomalous DA efflux (ADE) and in transgenic mice leads to changes in locomotor patterns, psychostimulant sensitivity, and impulsivity. Here, we show that the penetrance of Val559 ADE is dictated by region-specific differences in how presynaptic D 2 -type autoreceptors (D2ARs) constrain DA signaling, biasing phenotypic effects to dorsal striatal projections. The Val559 model illustrates how the impact of genetic variation underlying neuropsychiatric disorders can be shaped by the differential engagement of synaptic regulatory mechanisms., (Copyright © 2018 the authors 0270-6474/18/385303-11$15.00/0.)

Background: Dopamine (DA) is a critical neuromodulator in the retina. Disruption of retinal DA synthesis and signaling significantly attenuates light-adapted, electroretinogram (ERG) responses, as well as contrast sensitivity and acuity. As these measures can be detected noninvasively, they may provide opportunities to detect disease processes linked to perturbed DA signaling. Recently, we identified a rare, functional DA transporter (DAT, SLC6A3) coding substitution, Ala559Val, in subjects with attention-deficit/hyperactivity disorder (ADHD), demonstrating that DAT Val559 imparts anomalous DA efflux (ADE) with attendant physiological, pharmacological, and behavioral phenotypes. To understand the broader impact of ADE on ADHD, noninvasive measures sensitive to DAT reversal are needed., Methods: Here, we explored this question through ERG-based analysis of retinal light responses, as well as HPLC measurements of retinal DA in DAT Val559 mice., Results: Male mice homozygous (HOM) for the DAT Val559 variant demonstrated increased, light-adapted ERG b-wave amplitudes compared to wild type (WT) and heterozygous (HET) mice, whereas dark-adapted responses were indistinguishable across genotypes. The elevated amplitude of the photopic light responses in HOM mice could be mimicked in WT mice by applying D 1 and D 4 DA receptor agonists and suppressed in HOM mice by introducing D 4 antagonist, supporting elevated retinal DA signaling arising from ADE. Following the challenge with amphetamine, WT exhibited an increase in light-adapted response amplitudes, while HOM did not. Total retinal DA content was similar across genotypes. Interestingly, female DAT Val559 HOM animals revealed no significant difference in photopic ERG responses when compared with WT and HET littermates., Conclusions: These data reveal that noninvasive, in vivo evaluation of retinal responses to light can reveal physiological signatures of ADE, suggesting a possible approach to the segregation of neurobehavioral disorders based on the DAT-dependent control of DA signaling.

The neurotransmitter dopamine (DA) regulates multiple behaviors across phylogeny, with disrupted DA signaling in humans associated with addiction, attention-deficit/ hyperactivity disorder, schizophrenia, and Parkinson's disease. The DA transporter (DAT) imposes spatial and temporal limits on DA action, and provides for presynaptic DA recycling to replenish neurotransmitter pools. Molecular mechanisms that regulate DAT expression, trafficking, and function, particularly in vivo , remain poorly understood, though recent studies have implicated rho-linked pathways in psychostimulant action. To identify genes that dictate the ability of DAT to sustain normal levels of DA clearance, we pursued a forward genetic screen in Caenorhabditis elegans based on the phenotype swimming-induced paralysis (Swip), a paralytic behavior observed in hermaphrodite worms with loss-of-function dat-1 mutations. Here, we report the identity of swip-13 , which encodes a highly conserved ortholog of the human atypical MAP kinase ERK8. We present evidence that SWIP-13 acts presynaptically to insure adequate levels of surface DAT expression and DA clearance. Moreover, we provide in vitro and in vivo evidence supporting a conserved pathway involving SWIP-13/ERK8 activation of Rho GTPases that dictates DAT surface expression and function. SIGNIFICANCE STATEMENT Signaling by the neurotransmitter dopamine (DA) is tightly regulated by the DA transporter (DAT), insuring efficient DA clearance after release. Molecular networks that regulate DAT are poorly understood, particularly in vivo Using a forward genetic screen in the nematode Caenorhabditis elegans , we implicate the atypical mitogen activated protein kinase, SWIP-13, in DAT regulation. Moreover, we provide in vitro and in vivo evidence that SWIP-13, as well as its human counterpart ERK8, regulate DAT surface availability via the activation of Rho proteins. Our findings implicate a novel pathway that regulates DA synaptic availability and that may contribute to risk for disorders linked to perturbed DA signaling. Targeting this pathway may be of value in the development of therapeutics in such disorders., (Copyright © 2017 the authors 0270-6474/17/379288-17$15.00/0.)

Modulation of neurotransmission by the catecholamine dopamine (DA) is conserved across phylogeny. In the nematode Caenorhabditis elegans, excess DA signaling triggers Swimming-Induced Paralysis (Swip), a phenotype first described in animals with loss of function mutations in the presynaptic DA transporter (dat-1). Swip has proven to be a phenotype suitable for the identification of novel dat-1 mutations as well as the identification of novel genes that impact DA signaling. Pharmacological manipulations can also induce Swip, though the reagents employed to date lack specificity and potency, limiting their use in evaluation of dat-1 expression and function. Our lab previously established the mammalian norepinephrine transporter (NET) inhibitor nisoxetine to be a potent antagonist of DA uptake conferred by DAT-1 following heterologous expression. Here we demonstrate the ability of low (μM) concentrations of nisoxetine to trigger Swip within minutes of incubation, with paralysis dependent on DA release and signaling, and non-additive with Swip triggered by dat-1 deletion. Using nisoxetine in combination with genetic mutations that impact DA release, we further demonstrate the utility of the drug for demonstrating contributions of presynaptic DA receptors and ion channels to Swip. Together, these findings reveal nisoxetine as a powerful reagent for monitoring multiple dimensions of DA signaling in vivo, thus providing a new resource that can be used to evaluate contributions of dat-1 and other genes linked to DA signaling without the potential for compensations that attend constitutive genetic mutations., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Glial cells play a critical role in shaping neuronal development, structure, and function. In a screen for Caenorhabditis elegans mutants that display dopamine (DA)-dependent, Swimming-Induced Paralysis (Swip), we identified a novel gene, swip-10, the expression of which in glia is required to support normal swimming behavior. swip-10 mutants display reduced locomotion rates on plates, consistent with our findings of elevated rates of presynaptic DA vesicle fusion using fluorescence recovery after photobleaching. In addition, swip-10 mutants exhibit elevated DA neuron excitability upon contact with food, as detected by in vivo Ca(2+) monitoring, that can be rescued by glial expression of swip-10. Mammalian glia exert powerful control of neuronal excitability via transporter-dependent buffering of extracellular glutamate (Glu). Consistent with this idea, swip-10 paralysis was blunted in mutants deficient in either vesicular Glu release or Glu receptor expression and could be phenocopied by mutations that disrupt the function of plasma membrane Glu transporters, most noticeably glt-1, the ortholog of mammalian astrocytic GLT1 (EAAT2). swip-10 encodes a protein containing a highly conserved metallo-β-lactamase domain, within which our swip-10 mutations are located and where engineered mutations disrupt Swip rescue. Sequence alignments identify the CNS-expressed gene MBLAC1 as a putative mammalian ortholog. Together, our studies provide evidence of a novel pathway in glial cells regulated by swip-10 that limits DA neuron excitability, DA secretion, and DA-dependent behaviors through modulation of Glu signaling., (Copyright © 2015 the authors 0270-6474/15/359409-15$15.00/0.)

The neurotransmitter dopamine (DA) plays a critical role in CNS circuits that provide for attention, executive function, reward responses, motivation and movement. DA is inactivated by the cocaine- and amphetamine-sensitive DA transporter (DAT), a protein that also provides a pathway for non-vesicular DA release. After a brief review of DAT function and psychostimulant actions, we consider the importance DAT in relation to the distinct firing patterns of DA neurons that permit awareness of novelty and reward. Finally, we review recent efforts to gather direct support for DAT-linked disorders, with a specific focus on DAT mutations recently identified in subjects with ADHD., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Both dopamine and glutamate are critically involved in cognitive processes such as working memory. Astrocytes, which express dopamine receptors, are essential elements in the termination of glutamatergic signaling: the astrocytic glutamate transporter GLT-1 is responsible for > 90% of cortical glutamate uptake. The effect of dopamine depletion on glutamate transporters in the prefrontal cortex (PFC) remains unknown. In an effort to determine if astrocytes are a locus of cortical dopamine-glutamate interactions, we examined the effects of chronic dopamine denervation on PFC protein and mRNA levels of glutamate transporters. PFC dopamine denervation elicited a marked increase in GLT-1 protein levels, but had no effect on levels of other glutamate transporters; high-affinity glutamate transport was positively correlated with the extent of dopamine depletion. GLT-1 gene expression was not altered. Our data suggest that dopamine depletion may lead to post-translational modifications that result in increased expression and activity of GLT-1 in PFC astrocytes. The glutamate transporter GLT-1 is expressed by astrocytes, which also express dopamine receptors. Regulation of prefrontal cortical (PFC) GLT-1 potentially offers a novel treatment approach to the cognitive deficits of schizophrenia. Partial PFC dopamine deafferentation increased membrane expression of GLT-1 protein and glutamate uptake, but did not alter levels of the other two neocortical glutamate transporters, GLAST and EAAC1., (© 2014 International Society for Neurochemistry.)

Dopamine (DA) signaling in the central nervous system mediates the addictive capacities of multiple commonly abused substances, including cocaine, amphetamine, heroin and nicotine. The firing of DA neurons residing in the ventral tegmental area (VTA), and the release of DA by the projections of these neurons in the nucleus accumbens (NAc), is under tight control by cholinergic signaling mediated by nicotinic acetylcholine (ACh) receptors (nAChRs). The capacity for cholinergic signaling is dictated by the availability and activity of the presynaptic, high-affinity, choline transporter (CHT, SLC5A7) that acquires choline in an activity-dependent matter to sustain ACh synthesis. Here, we present evidence that a constitutive loss of CHT expression, mediated by genetic elimination of one copy of the Slc5a7 gene in mice (CHT+/-), leads to a significant reduction in basal extracellular DA levels in the NAc, as measured by in vivo microdialysis. Moreover, CHT heterozygosity results in blunted DA elevations following systemic nicotine or cocaine administration. These findings reinforce a critical role of ACh signaling capacity in both tonic and drug-modulated DA signaling and argue that genetically imposed reductions in CHT that lead to diminished DA signaling may lead to poor responses to reinforcing stimuli, possibly contributing to disorders linked to perturbed cholinergic signaling including depression and attention-deficit hyperactivity disorder (ADHD)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A subset of patients with Parkinson's disease acquires a debilitating dementia characterized by severe cognitive impairments (i.e. Parkinson's disease dementia; PDD). Brains from PDD patients show extensive cholinergic loss as well as dopamine (DA) depletion. We used a mutant mouse model to directly test whether combined cholinergic and DA depletion leads to a cognitive profile resembling PDD. Mice carrying heterozygous deletion of the high-affinity, hemicholinium-3-sensitive choline transporter (CHT(HET)) show reduced levels of acetylcholine throughout the brain. We achieved bilateral DA depletion in CHT(HET) and wild-type (WT) littermates via intra-striatal infusion of 6-hydroxydopamine (6-OHDA), or used vehicle as control. Executive function and memory were evaluated using rodent versions of cognitive tasks commonly used with human subjects: the set-shifting task and spatial and novel-object recognition paradigms. Our studies revealed impaired acquisition of attentional set in the set-shifting paradigm in WT-6OHDA and CHT(HET)-vehicle mice that was exacerbated in the CHT(HET)-6OHDA mice. The object recognition test following a 24-hour delay was also impaired in CHT(HET)-6OHDA mice compared with all other groups. Treatment with acetylcholinesterase (AChE) inhibitors physostigmine (0.05 or 0.1 mg/kg) and donepezil (0.1 and 0.3 mg/kg) reversed the impaired object recognition of the CHT(HET)-6OHDA mice. Our data demonstrate an exacerbated cognitive phenotype with dual ACh and DA depletion as compared with either insult alone, with traits analogous to those observed in PDD patients. The results suggest that combined loss of DA and ACh could be sufficient for pathogenesis of specific cognitive deficits in PDD.

Disrupted dopamine (DA) signaling is believed to contribute to the core features of multiple neuropsychiatric and neurodegenerative disorders. Essential features of DA neurotransmission are conserved in the nematode Caenorhabditis elegans, providing us with an opportunity to implement forward genetic approaches that may reveal novel, in vivo regulators of DA signaling. Previously, we identified a robust phenotype, termed Swimming-induced paralysis (Swip), that emerges in animals deficient in the plasma membrane DA transporter. Here, we report the use and quantitative analysis of Swip in the identification of mutant genes that control DA signaling. Two lines captured in our screen (vt21 and vt22) bear novel dat-1 alleles that disrupt expression and surface trafficking of transporter proteins in vitro and in vivo. Two additional lines, vt25 and vt29, lack transporter mutations but exhibit genetic, biochemical, and behavioral phenotypes consistent with distinct perturbations of DA signaling. Our studies validate the utility of the Swip screen, demonstrate the functional relevance of DA transporter structural elements, and reveal novel genomic loci that encode regulators of DA signaling.

The neurotransmitter dopamine (DA) modulates brain circuits involved in attention, reward, and motor activity. Synaptic DA homeostasis is primarily controlled via two presynaptic regulatory mechanisms, DA D(2) receptor (D(2)R)-mediated inhibition of DA synthesis and release, and DA transporter (DAT)-mediated DA clearance. D(2)Rs can physically associate with DAT and regulate DAT function, linking DA release and reuptake to a common mechanism. We have established that the attention-deficit hyperactivity disorder-associated human DAT coding variant Ala559Val (hDAT A559V) results in anomalous DA efflux (ADE) similar to that caused by amphetamine-like psychostimulants. Here, we show that tonic activation of D(2)R provides support for hDAT A559V-mediated ADE. We determine in hDAT A559V a pertussis toxin-sensitive, CaMKII-dependent phosphorylation mechanism that supports D(2)R-driven DA efflux. These studies identify a signaling network downstream of D(2)R activation, normally constraining DA action at synapses, that may be altered by DAT mutation to impact risk for DA-related disorders.

The Caenorhabditis elegans (C. elegans) dopamine (DA) transporter (DAT-1) regulates DA signaling through efficient DA reuptake following synaptic release. In addition to its DA transport function, DAT-1 generates detectable DA-gated currents that may influence neuronal excitability. Previously, we provided evidence that single Cl-channel events underlie DAT-1 currents. In these studies, we identified a distinct population of altered DAT-1 currents arising from DAT-1 transgenic constructs bearing an N-terminal GFP fusion. The presence of these channels suggested disruption of an endogenous regulatory mechanism that modulates occupancy of DAT-1 channel states. A leading candidate for such a regulator is the SNARE protein syntaxin 1A (Syn1A), previously found to interact with homologous transporters through N-terminal interactions. Here we establish that UNC-64 (C. elegans Syn1A homologue) associates with DAT-1 and suppresses transporter channel properties. In contrast, GFP::DAT-1 is unable to form stable transporter/UNC-64 complexes that limit channel states. Although DAT-1 and GFP::DAT-1 expressing DA neurons exhibit comparable DA uptake, GFP::DAT-1 animals exhibit swimming-induced paralysis (SWIP), a phenotype associated with excess synaptic DA release and spillover. We propose that loss of UNC-64/DAT-1 interactions leads to enhanced synaptic DA release, providing a novel mechanism for DA neuron sensitization that may be relevant to mechanisms of DA-associated disorders.

Dopamine (DA) signaling at synapses is tightly coordinated through opposing mechanisms of vesicular fusion-mediated DA release and transporter-mediated DA clearance. Altered brain DA signaling is suspected to underlie multiple brain disorders, including schizophrenia, Parkinson's disease, bipolar disorder, and attention-deficit hyperactivity disorder (ADHD). We identified a pedigree containing two male children diagnosed with ADHD who share a rare human DA transporter (DAT; SLC6A3) coding variant, Ala559Val. Among >1000 control and affected subjects, the Val559 variant has only been isolated once previously, in a female subject with bipolar disorder. Although hDAT Ala559Val supports normal DAT protein and cell surface expression, as well as normal DA uptake, the variant exhibits anomalous DA efflux from DA-loaded cells. We also demonstrate that hDAT Ala599Val exhibits increased sensitivity to intracellular Na(+), but not intracellular DA, and displays exaggerated DA efflux at depolarized potentials. Remarkably, the two most common ADHD medications, amphetamine and methylphenidate, both block hDAT Ala559Val-mediated DA efflux, whereas these drugs have opposite actions at wild-type hDAT. Our findings reveal that DA efflux, typically associated with amphetamine-like psychostimulants, can be produced through a heritable change in hDAT structure. Because multiple gene products are known to coordinate to support amphetamine-mediated DA efflux, the properties of hDAT Ala559Val may have broader significance in identifying a new mechanism through which DA signaling disorders arise. Additionally, they suggest that block of inappropriate neurotransmitter efflux may be an unsuspected mechanism supporting the therapeutic actions of existing transporter-directed medications.

The catecholamine dopamine (DA) functions as a powerful modulatory neurotransmitter in both invertebrates and vertebrates. As in man, DA neurons in the nematode Caenorhabditis elegans express a cocaine-sensitive transporter (DAT-1), presumably to regulate synaptic DA signaling and limit DA spillover to extrasynaptic sites, although evidence supporting this is currently lacking. In this report, we describe and validate a novel and readily quantifiable phenotype, swimming-induced paralysis (SWIP) that emerges in DAT-1-deficient nematodes when animals exert maximal physical activity in water. We verify the dependence of SWIP on DA biosynthesis, vesicular packaging, synaptic release, and on the DA receptor DOP-3. Using DAT-1 specific antibodies and GFP::DAT-1 fusions, we demonstrate a synaptic enrichment of DAT-1 that is achieved independently of synaptic targeting of the vesicular monoamine transporter (VMAT). Importantly, dat-1 deletions and point mutations that disrupt DA uptake in cultured C. elegans neurons and/or impact DAT-1 synaptic localization in vivo generate SWIP. SWIP assays, along with in vivo imaging of wild-type and mutant GFP::DAT-1 fusions identify a distal COOH terminal segment of the transporter as essential for efficient somatic export, synaptic localization and in vivo DA clearance. Our studies provide the first description of behavioral perturbations arising from altered trafficking of DATs in vivo in any organism and support a model whereby endogenous DA actions in C. elegans are tightly regulated by synaptic DAT-1.

The behavioral effects of psychomotor stimulants such as amphetamine (AMPH) arise from their ability to elicit increases in extracellular dopamine (DA). These AMPH-induced increases are achieved by DA transporter (DAT)-mediated transmitter efflux. Recently, we have shown that AMPH self-administration is reduced in rats that have been depleted of insulin with the diabetogenic agent streptozotocin (STZ). In vitro studies suggest that hypoinsulinemia may regulate the actions of AMPH by inhibiting the insulin downstream effectors phosphotidylinositol 3-kinase (PI3K) and protein kinase B (PKB, or Akt), which we have previously shown are able to fine-tune DAT cell-surface expression. Here, we demonstrate that striatal Akt function, as well as DAT cell-surface expression, are significantly reduced by STZ. In addition, our data show that the release of DA, determined by high-speed chronoamperometry (HSCA) in the striatum, in response to AMPH, is severely impaired in these insulin-deficient rats. Importantly, selective inhibition of PI3K with LY294002 within the striatum results in a profound reduction in the subsequent potential for AMPH to evoke DA efflux. Consistent with our biochemical and in vivo electrochemical data, findings from functional magnetic resonance imaging experiments reveal that the ability of AMPH to elicit positive blood oxygen level-dependent signal changes in the striatum is significantly blunted in STZ-treated rats. Finally, local infusion of insulin into the striatum of STZ-treated animals significantly recovers the ability of AMPH to stimulate DA release as measured by high-speed chronoamperometry. The present studies establish that PI3K signaling regulates the neurochemical actions of AMPH-like psychomotor stimulants. These data suggest that insulin signaling pathways may represent a novel mechanism for regulating DA transmission, one which may be targeted for the treatment of AMPH abuse and potentially other dopaminergic disorders.

Aims: In this review, we highlight the identification and analysis of molecules orchestrating dopamine (DA) signaling in the nematode Caenorhabditis elegans, focusing on recent characterizations of DA transporters and receptors., Methods: We illustrate the isolation and characterization of molecules important for C. elegans DA synthesis, packaging, reuptake and signaling and examine how mutations in these proteins are being exploited through in vitro and in vivo paradigms to yield novel insights of protein structure, DA signaling pathways and DA-supported behaviors., Results: DA signaling in the worm, as in man, arises by synaptic and nonsynaptic release from a small number of cells that exert modulatory control over a larger network underlying C. elegans behavior., Conclusions: The C. elegans model system offers unique opportunities to elucidate ill-defined pathways that support DA release, inactivation, and signaling in addition to clarifying mechanisms of DA-mediated behavioral plasticity. Further use of the model offers prospects for the identification of novel genes and proteins whose study may yield benefits for DA-supported neural disorders in man.

The presynaptic dopamine (DA) transporter (DAT) is a major determinant of synaptic DA inactivation, an important target for psychostimulants including cocaine and amphetamine, and a mediator of DA neuron vulnerability to the neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion. To exploit genetic approaches for the study of DATs and neural degeneration, we exploited the visibility of green fluorescent protein (GFP)-tagged DA neurons in transgenic nematodes to implement a forward genetic screen for suppressors of 6-OHDA sensitivity. In our initial effort, we identified three novel dat-1 alleles conferring 6-OHDA resistance. Two of the dat-1 alleles derive from point mutations in conserved glycine residues (G55, G90) in contiguous DAT-1 transmembrane domains (TM1 and TM2, respectively), whereas the third allele results in altered translation of the transporter's COOH terminus. Our studies reveal biosynthetic, trafficking and functional defects in the DAT-1 mutants, exhibited both in vitro and in vivo. These studies validate a forward genetic approach to the isolation of DA neuron-specific toxin suppressors and point to critical contributions of the mutated residues, as well as elements of the DAT-1 COOH terminus, to functional expression of catecholamine transporters in neurons.

Drugs of abuse, including cocaine, amphetamine (AMPH), and heroin, elevate extracellular dopamine (DA) levels in the brain, thereby altering the activity/plasticity of reward circuits and precipitating addiction. The physiological release of DA occurs through the calcium-dependent fusion of a synaptic vesicle with the plasma membrane. Extracellular DA is cleared by uptake through the Na+/Cl- -dependent DA transporter (DAT). In contrast, the substrate AMPH induces nonvesicular release of DA mediated by DAT. Extracellular AMPH is generally believed to trigger DA efflux through DAT by facilitating exchange for cytosolic DA. Here, in outside-out patches from heterologous cells stably expressing DAT or from dopaminergic neurons, by using ionic conditions in the patch pipette that mimic those produced by AMPH stimulation, we report that AMPH causes DAT-mediated DA efflux by two independent mechanisms: (i) a slow process consistent with an exchange mechanism and (ii) a process that results in rapid (millisecond) bursts of DA efflux through a channel-like mode of DAT. Because channel-like release of DA induced by AMPH is rapid and contains a large number of DA molecules, with a single burst of DA on par with a quantum of DA from exocytotic release of a vesicle, this burst mode of release may play a role in the synaptic actions and psychostimulant properties of AMPH and related compounds. Unlike AMPH, the endogenous substrate DA, when present on both sides of the plasma membrane, inhibits this channel-like activity, thereby suggesting that the DAT channel-like mode cannot accumulate DA against a concentration gradient.

The norepinephrine (NE) system is implicated in the etiology and treatment of depression and drugs blocking the NE transporter (NET) are effective antidepressants. It is possible that dopamine (DA) also plays a role in depression and the action of antidepressant drugs, although the mechanisms whereby NE and DA interact have not been fully elaborated. We examined whether NE neurons might alter DA transmission via synaptic projections to cells in the ventral tegmental area (VTA) by using electron microscopic dual labeling immunocytochemistry in the rat and monkey. NET was used as a marker for NE axons, whereas the catecholamine synthetic enzyme, tyrosine hydroxylase (TH), served as a label for DA neurons. We observed three types of spatial relationships between these profiles that were similar in type but varied in frequency between rodent and primate. The most common arrangement involved nonsynaptic appositions between NET-immunoreactive (-ir) axons and TH-ir dendrites. Such relationships may facilitate extrasynaptic actions of NE on DA cell activity. The other commonly observed arrangement involved adjacent profiles that were otherwise separated by glia. These relationships may represent regions where NE is prevented from reaching DA cells. In only a few cases were synapses observed between NET-ir axons and TH-ir dendrites. This finding suggests that NE can synaptically regulate DA neurons, although functional interactions are more likely to involve extrasynaptic mechanisms. Finally, in the VTA of both species the majority of NET-ir axons exhibited no detectable TH immunoreactivity. The latter finding agrees with observations in cortical regions and represents the first report of its type in a subcortical structure., (Copyright 2004 Wiley-Liss, Inc.)

The prefrontal cortex (PFC) is a likely site of action for the therapeutic efficacy of antidepressants that inhibit norepinephrine (NE) reuptake. Moreover, drugs that block the NE transporter (NET) increase extracellular levels of both NE and dopamine (DA), an interaction that may contribute to their therapeutic properties. To examine the subcellular localization of NET and to investigate the spatial relationships between presumed NE and DA axons within the rat prelimbic PFC, we combined immunogold-silver localization of NET with immunoperoxidase staining for the catecholamine synthetic enzyme tyrosine hydroxylase (TH). An additional aim was to quantify the proportion of profiles dually labeled for NET and TH to test the common observation that TH immunolabeling is relatively selective for DA axons. NET-immunoreactive (NET-ir) axonal profiles were typically unmyelinated and occasionally were observed to form symmetric axodendritic synapses. The majority of immunogold NET labeling was unexpectedly observed in the cytoplasm rather than on the plasma membrane. Furthermore, in tissue dually labeled for both NET and TH, only 8-10% of profiles contained both markers. Unlike observations for singly labeled profiles, gold-silver particles for NET in dually labeled axons were localized primarily to the plasmalemma. A systematic survey of terminals labeled only for TH revealed that they were typically separated by at least 1.2 mum from NET-ir varicosities, and the two profile types were not seen to contact common targets. These results suggest that, in the rat PFC, NE axons (1) contain predominantly cytoplasmic NET, (2) infrequently contain TH immunolabeling, and (3) may interact with probable DA afferents by means of extrasynaptic mechanisms., (Copyright 2003 Wiley-Liss, Inc.)

Overexpression of human alpha-synuclein in model systems, including cultured neurons, drosophila and mice, leads to biochemical and pathological changes that mimic synucleopathies including Parkinson's disease. We have overexpressed both wild-type (WT) and mutant alanine53-->threonine (A53T) human alpha-synuclein by transgenic injection into Caenorhabditis elegans. Motor deficits were observed when either WT or A53T alpha-synuclein was overexpressed with a pan-neuronal or motor neuron promoter. Neuronal and dendritic loss were accelerated in all three sets of C. elegans dopaminergic neurons when human alpha-synuclein was overexpressed under the control of a dopaminergic neuron or pan-neuronal promoter, but not with a motor neuron promoter. There were no significant differences in neuronal loss between overexpressed WT and A53T forms or between worms of different ages (4 days, 10 days or 2 weeks). These results demonstrate neuronal and behavioral perturbations elicited by human alpha-synuclein in C. elegans that are dependent upon expression in specific neuron subtypes. This transgenic model in C. elegans, an invertebrate organism with excellent experimental resources for further genetic manipulation, may help facilitate dissection of pathophysiologic mechanisms of various synucleopathies.

The neurotransmitter dopamine (DA) plays a central role in the coordination of movement, attention, and the recognition of reward. Loss of DA from the basal ganglia, as a consequence of degeneration of neurons in the substantia nigra, triggers postural instability and Parkinson's disease (PD). DA transporters (DATs) regulate synaptic DA availability and provide a conduit for the uptake of DA mimetic neurotoxins, which can be used to evoke neuronal death and Parkinson-like syndrome. Recently, we have explored the sensitivity of DA neurons in the nematode Caenorhabditis elegans to the Parkinsonian-inducing neurotoxin 6-hydroxydopamine (6-OHDA) and found striking similarities, including DAT dependence, to neurodegeneration observed in mammalian models. In this review, we present our findings in the context of molecular and behavioral dimensions of DA signaling in C. elegans with an eye toward opportunities for uncovering DAT mutants, DAT regulators, and components of toxin-mediated cell death.

Parkinson's disease is a complex neurodegenerative disorder characterized by the death of brain dopamine neurons. In mammals, dopamine neuronal degeneration can be triggered through exposure to neurotoxins accumulated by the presynaptic dopamine transporter (DAT), including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium. We have established a system for the pharmacological and genetic evaluation of neurotoxin-induced dopamine neuronal death in Caenorhabditis elegans. Brief (1 h) exposure of green fluorescent protein-tagged, living worms to 6-OHDA causes selective degeneration of dopamine neurons. We demonstrate that agents that interfere with DAT function protect against 6-OHDA toxicity. 6-OHDA-triggered neural degeneration does not require the CED-3/CED-4 cell death pathway, but is abolished by the genetic disruption of the C. elegans DAT.

Aberrant dopamine (DA) signalling has been implicated in various neuropsychiatric disorders, including attention‐deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), schizophrenia, bipolar disorder (BPD) and addiction. The availability of extracellular DA is sculpted by the exocytotic release of vesicular DA and subsequent transporter‐mediated clearance, rendering the presynaptic DA transporter (DAT) a crucial regulator of DA neurotransmission. D2‐type DA autoreceptors (D2ARs) regulate multiple aspects of DA homeostasis, including (i) DA synthesis, (ii) vesicular release, (iii) DA neuron firing and (iv) the surface expression of DAT and DAT‐mediated DA clearance. The DAT Val559 variant, identified in boys with ADHD or ASD, as well as in a girl with BPD, supports anomalous DA efflux (ADE), which we have shown drives tonic activation of D2ARs. Through ex vivo and in vivo studies of the DAT Val559 variant using transgenic knock‐in mice, we have uncovered a circuit and sex‐specific capacity of D2ARs to regulate DAT, which consequently disrupts DA signalling and behaviour differently in males and females. Our studies reveal the ability of the construct‐valid DAT Val559 model to elucidate endogenous mechanisms that support DA signalling, findings that may be of translational and/or therapeutic importance. [ABSTRACT FROM AUTHOR]

Abstract The catecholamine neurotransmitter dopamine (DA) exerts powerful modulatory control of physiology and behavior across phylogeny. Perturbations of DA signaling in humans are associated with multiple neurodegenerative and behavioral disorders, including Parkinson's disease, attention-deficit/hyperactivity disorder, addiction and schizophrenia. In the nematode C. elegans , DA signaling regulates mating behavior, learning, food seeking and locomotion. Previously, we demonstrated that loss of function mutations in the dat-1 gene that encodes the presynaptic DA transporter (DAT-1) results in a rapid cessation of movement when animals are placed in water, termed Swimming Induced Paralysis (Swip). Loss of function mutations in genes that support DA biosynthesis, DA vesicular packaging and DA action at the extrasynaptic D2-type DA receptor DOP-3 suppress Swip in dat-1 animals, consistent with paralysis as arising from excessive DA signaling. Although animals grown on the vesicular monoamine transporter antagonist reserpine diminish Swip, the drug must be applied chronically, can impact the signaling of multiple biogenic amines, and has been reported to have penetrant, off-target actions. Here, we demonstrate that the antipsychotic drug azaperone potently and rapidly suppresses Swip behavior in either dat-1 mutants, as well as in wildtype animals treated with the DAT-1 antagonist nisoxetine, with genetic experiments consistent with DOP-3 antagonism as the mechanism of Swip suppression. Reversal of Swip in previously paralyzed dat-1 animals by azaperone application demonstrates an otherwise functionally-intact swimming circuit in these mutants. Finally, whereas azaperone suppresses DA-dependent Swip, the drug fails to attenuate the DA-independent paralysis induced by βPEA, aldicarb or genetic disruption of γ-aminobutyric acid (GABA) signaling. We discuss our findings with respect to the use of azaperone as a potent and selective tool in the identification and analysis of presynaptic mechanisms that regulate DA signaling. Highlights • The antipsychotic azaperone suppresses swimming-induced paralysis (Swip) in C. elegans. • Azaperone effects on Swip are specific to paralysis driven by excess DA signaling. • Azaperone suppression of Swip involves blockade of D2-type DOP-3 DA receptors. • Azaperone restores swimming behavior to animals paralyzed by excess DOP-3 stimulation. • Ongoing DA receptor signaling drives Swip in dat-1 animals, not circuit compensations. [ABSTRACT FROM AUTHOR]

Disruptions of dopamine(DA) signaling contribute to a broad spectrum of neuropsychiatric disorders, including attention-deficit hyperactivity disorder (ADHD), addiction, bipolar disorder, and schizophrenia. Despite evidence that risk for these disorders derives from heritable variation in DA-linked genes, a better understanding is needed of the molecular and circuit context through which gene variation drives distinct disease traits. Previously, we identified the DA transporter (DAT) variant Val559 in subjects with ADHD and established that the mutation supports anomalous DAT-mediated DA efflux (ADE). Here, we demonstrate that region-specific contributions of D2 autoreceptors (D2AR) to presynaptic DA homeostasis dictate the consequences of Val559 expression in adolescent male mice. We show that activation of D2ARs in the WT dorsal striatum (DS), but not ventral striatum (VS), increases DAT phosphorylation and surface trafficking. In contrast, the activity of tyrosine hydroxylase (TH) is D2AR-dependentinbothregions. In the DS but not VS of Val559 mice, tonic activation ofD2ARsdrivesapositive feedback loop that promotes surface expression of efflux-prone DATs, raising extracellular DA levels and overwhelming DAT-mediated DA clearance capacity. WhereasD2ARsthat regulate DAT are tonically activated in the Val559 DS,D2ARsthat regulate TH become desensitized, allowing maintenance of cytosolic DA needed to sustain ADE. Together with prior findings, our results argue for distinctD2ARpools that regulate DA synthesis versus DA release and inactivation and offer a clear example of how the penetrance of gene variation can be limited to a subset of expression sites based on differences in intersecting regulatory networks. [ABSTRACT FROM AUTHOR]

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 presynaptic and postsynaptic 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 hDAT (human DAT) coding variant, R615C, located in the distal C terminus of the transporter, a region previously implicated in constitutive and regulated transporter trafficking. Here, we demonstrate that, whereas wild-type DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively and demonstrate insensitivity to the endocytic effects of AMPH and PKC (protein kinase C) 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 CaMKII (calcium/calmodulin-dependent protein kinase II) and flotillin-1 interactions. Using C-terminal peptides derived from wild-type DAT and the R615C variant, we establish that the DAT 615C C terminus can act dominantly to preclude AMPHregulation of wild-type DAT. Mutagenesis of DATC-terminal sequences suggests 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. [ABSTRACT FROM AUTHOR]

The neurotransmitter dopamine (DA) modulates brain circuits involved in attention, reward, and motor activity. Synaptic DA homeostasis is primarily controlled via two presynaptic regulatory mechanisms, DA D2 receptor (D2R)-mediated inhibition of DA synthesis and release, and DA transporter (DAT)-mediated DA clearance. D2Rs can physically associate with DAT and regulate DAT function, linking DA release and reuptake to a common mechanism. We have established that the attention-deficit hyperactivity disorder-associated human DAT coding variant Ala559Val (hDAT A559V) results in anomalous DA efflux (ADE) similar to that caused by amphetamine-like psychostimulants. Here, we show that tonic activation of D2R provides support for hDAT A559V-mediated ADE. We determine in hDAT A559V a pertussis toxin-sensitive, CaMKII-dependent phosphorylation mechanism that supports D2R-driven DA efflux. These studies identify a signaling network downstream of D2R activation, normally constraining DA action at synapses, that may be altered by DAT mutation to impact risk for DA-related disorders. [ABSTRACT FROM AUTHOR]

Background: Tegaserod is a novel drug for the treatment of constipation-predominant irritable bowel syndrome. Tegaserod is thought to exert its prokinetic effect as a selective partial agonist of serotonin receptor type 4 (5-HT4) receptors located in the enteric nervous system. It is unknown, however, whether tegaserod interacts with the human serotonin reuptake transporter (hSERT) and the uptake transporters for dopamine (hDAT) and norepinephrine (hNET). Therefore, the aim of the present study was to investigate whether tegaserod inhibits SERT-, DAT-, and NET-mediated transport. Methods: Tegaserod inhibition of SERT-mediated [3H]5-HT and NET- and DAT-mediated [3H]dopamine uptake was measured in human embryonic kidney (HEK) 293 cells stably expressing hSERT, hDAT, and hNET in comparison with untransfected control HEK293-FT cells. Results: Tegaserod inhibited SERT-, DAT-, and NET-mediated transport with IC50-values of 11.7, 20.7, and 3.2 μmol/l, respectively, while 100 μmol/l estrone-3-sulfate or taurocholic acid, used as negative controls, failed to inhibit hSERT-mediated transport. Using Dixon plot analysis, inhibition kinetics yielded a non-competitive type of inhibition with an apparent inhibition konstant (Ki) of 3.1 μmol/l for SERT-mediated 5-HT transport. Conclusion: In the present study we propose an additional mechanism of action for tegaserod as a serotonin uptake inhibitor. By inhibiting SERT and increasing local 5-HT concentrations in the gut wall, tegaserod might exert its prokinetic action via a synergism between 5-HT4 agonism and low-affinity SERT inhibition. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Multiple, rare, human dopamine (DA) transporter (hDAT, SLC6A3) coding variants have been described, though to date they have been incompletely characterized. Here we present studies analyzing the function and regulation of five naturally occurring coding variants, V55A, R237Q, V382A, A559V and E602G, expressed in COS-7 and SH-SY5Y cells. All variants, except V382A, exhibited levels of surface protein expression and DA transport activity comparable to hDAT. V382A, divergent at the most highly conserved residue among reported hDAT variants, exhibited significantly diminished surface expression, likely derived from inefficient plasma membrane delivery. Moreover, a greater expression of V382A protein was required to achieve comparable levels of transport to hDAT, consistent with a loss of transport function. V382A displayed a decrease in sensitivity to phorbol ester (PMA)-induced internalization, as well as an altered substrate selectivity for DA versus norepinephrine (NE). Analysis of PMA-induced V382A internalization revealed a trafficking-independent action of PMA, consistent with the existence of a surface-localized, transport-inactive state. [Copyright &y& Elsevier]

Focuses on the clinical importance of dopamine. Significance of dopamine as a neurotransmitter in brain neural circuits in carrying information about movement; Implications of the death of dopaminergic neurons for the occurrence of Parkinson's disease (PD); Use of imaging dopamine transporters on recording the progression of PD.

Abstract: Compounds capable of inhibiting the dopamine transporter protein (DAT) that can be conjugated to cadmium selenide/zinc sulfide/core shell nanocrystals may be used to image the location and distribution of the DAT in neuronal cell membranes. This letter describes the synthesis of biotinylated analogs of the DAT antagonists GBR 12909 and GBR 12935 that can be attached to streptavidin coated cadmium selenide/zinc sulfide/core shell nanocrystals. [Copyright &y& Elsevier]

Recent genetic analyses have provided evidence that clinical commonalities associated with different psychiatric diagnoses often have shared mechanistic underpinnings. The development of animal models expressing functional genetic variation attributed to multiple disorders offers a salient opportunity to capture molecular, circuit and behavioral alterations underlying this hypothesis. In keeping with studies suggesting dopaminergic contributions to attention-deficit hyperactivity disorder (ADHD), bipolar disorder (BPD) and autism spectrum disorder (ASD), subjects with these diagnoses have been found to express a rare, functional coding substitution in the dopamine (DA) transporter (DAT), Ala559Val. We developed DAT Val559 knock-in mice as a construct valid model of dopaminergic alterations that drive multiple clinical phenotypes, and here evaluate the impact of lifelong expression of the variant on impulsivity and motivation utilizing the 5- choice serial reaction time task (5-CSRTT) and Go/NoGo as well as tests of time estimation (peak interval analysis), reward salience (sucrose preference), and motivation (progressive ratio test). Our findings indicate that the DAT Val559 variant induces impulsivity behaviors that are dependent upon the reward context, with increased impulsive action observed when mice are required to delay responding for a reward, whereas mice are able to withhold responding if there is a probability of reward for a correct rejection. Utilizing peak interval and progressive ratio tests, we provide evidence that impulsivity is likely driven by an enhanced motivational phenotype that also may drive faster task acquisition in operant tasks. These data provide critical validation that DAT, and more generally, DA signaling perturbations can drive impulsivity that can manifest in specific contexts and not others, and may rely on motivational alterations, which may also drive increased maladaptive reward seeking. [ABSTRACT FROM AUTHOR]

The monoamine neurotransmitter dopamine (DA) acts across phylogeny to modulate both simple and complex behaviors. The presynaptic DA transporter (DAT) is a major determinant of DA signaling capacity in ensuring efficient extracellular DA clearance. In humans, DAT is also a major target for prescribed and abused psychostimulants. Multiple structural determinants of DAT function and regulation have been defined, though largely these findings have arisen from heterologous expression or ex vivo cell culture studies. Loss of function mutations in the gene encoding the Caenhorhabditis elegans DAT ( dat - 1 ) produces rapid immobility when animals are placed in water, a phenotype termed swimming-induced paralysis (Swip). The ability of a DA neuron-expressed, GFP-tagged DAT-1 fusion protein (GFP::DAT-1) to localize to synapses and rescue Swip in these animals provides a facile approach to define sequences supporting DAT somatic export and function in vivo . In prior studies, we found that truncation of the last 25 amino acids of the DAT-1 C-terminus (Δ25) precludes Swip rescue, supported by a deficit in GFP::DAT-1 synaptic localization. Here, we further defined the elements within Δ25 required for DAT-1 export and function in vivo . We identified two conserved motifs ( 584 KW 585 and 591 PYRKR 595 ) where mutation results in a failure of GFP::DAT-1 to be efficiently exported to synapses and restore DAT-1 function. The 584 KW 585 motif conforms to a sequence proposed to support SEC24 binding, ER export from the endoplasmic reticulum (ER), and surface expression of mammalian DAT proteins, whereas the 591 PYRKR 595 sequence conforms to a 3R motif identified as a SEC24 binding site in vertebrate G-protein coupled receptors. Consistent with a potential role of SEC24 orthologs in DAT-1 export, we demonstrated DA neuron-specific expression of a sec-24.2 transcriptional reporter. Mutations of the orthologous C-terminal sequences in human DAT (hDAT) significantly reduced transporter surface expression and DA uptake, despite normal hDAT protein expression. Although, hDAT mutants retained SEC24 interactions, as defined in co-immunoprecipitation studies. However, these mutations disrupted the ability of SEC24D to enhance hDAT surface expression. Our studies document an essential role of conserved DAT C-terminal sequences in transporter somatic export and synaptic localization in vivo , that add further support for important roles for SEC24 family members in efficient transporter trafficking. [ABSTRACT FROM AUTHOR]

Abstract: Background: The nematode Caenhorhabditis elegans offers great power for the identification and characterization of genes that regulate behavior. In support of this effort, analytical methods are required that provide dimensional analyses of subcomponents of behavior. Previously, we demonstrated that loss of the presynaptic dopamine (DA) transporter, dat-1, evokes DA-dependent Swimming-Induced Paralysis (Swip) (Mcdonald et al., 2007), a behavior compatible with forward genetic screens (Hardaway et al., 2012). New method: Here, we detail the development and implementation of SwimR, a set of tools that provide for an automated, kinetic analysis of C. elegans Swip. SwimR relies on open source programs that can be freely implemented and modified. Results: We show that SwimR can display time-dependent alterations of swimming behavior induced by drug-treatment, illustrating this capacity with the dat-1 blocker and tricyclic antidepressant imipramine (IMI). We demonstrate the capacity of SwimR to extract multiple kinetic parameters that are impractical to obtain in manual assays. Comparison with existing methods: Standard measurements of C. elegans swimming utilizes manual assessments of the number of animals exhibiting swimming versus paralysis. Our approach deconstructs the time course and rates of movement in an automated fashion, offering a significant increase in the information that can be obtained from swimming behavior. Conclusions: The SwimR platform is a powerful tool for the deconstruction of worm thrashing behavior in the context of both genetic and pharmacological manipulations that can be used to segregate pathways that underlie nematode swimming mechanics. [Copyright &y& Elsevier]

Highlights: [•] We review current evidence for altered DA signaling and DAT function in ADHD. [•] We describe the discovery of ADHD-associated, rare functional genetic variation. [•] We describe the creation of the DAT Val559 mouse, a construct valid model of ADHD. [Copyright &y& Elsevier]

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