Dopamine Efflux Research Articles

Region-Specific Regulation of Presynaptic Dopamine Homeostasis by D2 Autoreceptors Shapes the In Vivo Impact of the Neuropsychiatric Disease-Associated DAT Variant Val559.

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-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 D2-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.

Brief exposure to obesogenic diet disrupts brain dopamine networks.

ObjectiveWe have previously demonstrated that insulin signaling, through the downstream signaling kinase Akt, is a potent modulator of dopamine transporter (DAT) activity, which fine-tunes dopamine (DA) signaling at the synapse. This suggests a mechanism by which impaired neuronal insulin receptor signaling, a hallmark of diet-induced obesity, may contribute to impaired DA transmission. We tested whether a short-term (two-week) obesogenic high-fat (HF) diet could reduce striatal Akt activity, a marker of central insulin, receptor signaling and blunt striatal and dopaminergic network responsiveness to amphetamine (AMPH).MethodsWe examined the effects of a two-week HF diet on striatal DAT activity in rats, using AMPH as a probe in a functional magnetic resonance imaging (fMRI) assay, and mapped the disruption in AMPH-evoked functional connectivity between key dopaminergic targets and their projection areas using correlation and permutation analyses. We used phosphorylation of the Akt substrate GSK3α in striatal extracts as a measure of insulin receptor signaling. Finally, we confirmed the impact of HF diet on striatal DA D2 receptor (D2R) availability using [18F]fallypride positron emission tomography (PET).ResultsWe found that rats fed a HF diet for only two weeks have reductions in striatal Akt activity, a marker of decreased striatal insulin receptor signaling and blunted striatal responsiveness to AMPH. HF feeding also reduced interactions between elements of the mesolimbic (nucleus accumbens–anterior cingulate) and sensorimotor circuits (caudate/putamen–thalamus–sensorimotor cortex) implicated in hedonic feeding. D2R availability was reduced in HF-fed animals.ConclusionThese studies support the hypothesis that central insulin signaling and dopaminergic neurotransmission are already altered after short-term HF feeding. Because AMPH induces DA efflux and brain activation, in large part via DAT, these findings suggest that blunted central nervous system insulin receptor signaling through a HF diet can impair DA homeostasis, thereby disrupting cognitive and reward circuitry involved in the regulation of hedonic feeding.

Mechanistic Insights into the Stimulant Properties of Novel Psychoactive Substances (NPS) and Their Discrimination by the Dopamine Transporter-In Silico and In Vitro Exploration of Dissociative Diarylethylamines.

Novel psychoactive substances (NPS) may have unsuspected addiction potential through possessing stimulant properties. Stimulants normally act at the dopamine transporter (DAT) and thus increase dopamine (DA) availability in the brain, including nucleus accumbens, within the reward and addiction pathway. This paper aims to assess DAT responses to dissociative diarylethylamine NPS by means of in vitro and in silico approaches. We compared diphenidine (DPH) and 2-methoxydiphenidine (methoxphenidine, 2-MXP/MXP) for their binding to rat DAT, using autoradiography assessment of [125I]RTI-121 displacement in rat striatal sections. The drugs’ effects on electrically-evoked DA efflux were measured by means of fast cyclic voltammetry in rat accumbens slices. Computational modeling, molecular dynamics and alchemical free energy simulations were used to analyse the atomistic changes within DAT in response to each of the five dissociatives: DPH, 2-MXP, 3-MXP, 4-MXP and 2-Cl-DPH, and to calculate their relative binding free energy. DPH increased DA efflux as a result of its binding to DAT, whereas MXP had no significant effect on either DAT binding or evoked DA efflux. Our computational findings corroborate the above and explain the conformational responses and atomistic processes within DAT during its interactions with the dissociative NPS. We suggest DPH can have addictive liability, unlike MXP, despite the chemical similarities of these two NPS.

Neurotoxic Dopamine Transporter Substrates Differentially Affect DAT Regulation, Uptake, and Reverse Transport

Synaptic action of the neurotransmitter dopamine (DA) is primarily terminated by reuptake into presynaptic dopaminergic neurons via dopamine transporters (DATs). DATs are integral plasmalemmal proteins whose reuptake function is tightly coordinated by interplay between the post translational modifications phosphorylation and palmitoylation. The psychostimulant amphetamine (AMPH) is a DAT substrate known to empty sequestered DA from vesicles and induce protein kinase C (PKC)‐dependent DAT phosphorylation, resulting in reverse transport of DA (efflux) greatly increased DA signaling. Elevated DA concentrations similar to those produced by AMPH create neurotoxic conditions as DA oxidation produces free radicals and hinders mitochondrial respiration, thus strict spatial and temporal control of cytosolic and synaptic DA is essential for neuronal health. DA induced oxidative stress is an important pathogenic mediator in Parkinson's disease (PD), and animal models of PD are generated by the neurotoxins 1‐methyl‐4‐phenylpyridinium ion (MPP+) and 6‐hydroxydopamine (6OHDA) which selectively target DAT‐expressing neurons, are translocated to the cytosol by DAT, and perturb the oxidative balance within the cell, producing nigrostriatal DA neuron death observed in PD. It is therefore intriguing to investigate DAT‐involved mechanisms in terms of PD etiology as atypical DAT function could contribute to DA oxidative stress and is hypothesized to play a role in other neurological disorders including attention deficit/hyperactivity disorder (ADHD) and addiction. DAT expression occurs around synaptic terminals, along axons, and on dendritic spines, and efflux via DATs located on dendrites in the substantia nigra has been shown to diminish dopaminergic signaling. It is possible, therefore, for aberrant regulation and localization of DA clearance and DAT‐mediated DA efflux to profoundly influence the excitability of neurons and play a significant role in neurodegeneration in the progression of PD. This is even more interesting in the light of recent studies revealing DAT polymorphisms in patients diagnosed with a psychiatric disorder or early onset PD that display an elevated basal DA efflux phenotype. In this study, we are investigating the effect of DAT substrates and PD‐inducing neurotoxins on DAT phosphorylation, palmitoylation, down‐regulation, and reverse transport to better understand aberrant DAT mechanisms which may contribute the onset of PD. Our findings thus far indicate MPP+ and 6OHDA differentially alter DAT function. 6OHDA inhibits basal and attenuates PMA‐induced DAT phosphorylation, does not significantly reduce DAT function, and decreases DA efflux. MPP+ does not change basal or PMA stimulated DAT phosphorylation yet it induces significant DA efflux which may be an additional means by which MPP+ produces neurodegeneration. This MPP+ induced efflux was not blunted by pretreatment with the PKC inhibitor bisindolylmaleimide (BIM). Although AMPH‐induced efflux was blunted after BIM treatment, significant efflux remained suggesting mechanisms independent of PKC mediated DAT phosphorylation for the stimulation of DA efflux.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research Resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphatidylinositol (4, 5)‐bisphosphate coordinates functional interactions in the dopamine transporter to promote amphetamine behaviors

The psychostimulant amphetamine (AMPH) mainly mediates its pharmacological and behavioral effects by increasing extracellular dopamine (DA) availability. DA homeostasis is maintained by the dopamine transporter (DAT), a presynaptic membrane protein that mediates the high‐affinity reuptake of released DA from the synaptic cleft. We have previously demonstrated that AMPH induces N‐term phosphorylation of the DAT, which leads to transport‐mediated efflux of DA. Furthermore, we have shown that phosphatidylinositol (4, 5)‐bisphosphate (PIP2) directly interacts with the DAT and facilitates AMPH‐induced DA efflux, but is not required for DA uptake. Specially, PIP2 binds DAT through electrostatic interactions with positively charged DAT N‐terminal residues. Disrupting the interactions between DAT and PIP2 or depleting PIP2 diminishes reverse transport (efflux) of DA. Previous studies on the human serotonin transporter show that non‐N‐terminal PIP2 binding sites (within the fourth intracellular loop) are essential for AMPH‐induced reverse transport of serotonin, but not substrate uptake. Together, these data led us to hypothesize that PIP2 coordinates functional interactions between the N‐terminus and the fourth intracellular loop to promote reverse transport of DA. Here, we demonstrate that depletion of PIP2 after DAT N‐terminus phosphorylation does not affect AMPH‐induced DA efflux, suggesting that N‐terminus phosphorylation is the limiting step of reverse transport of DA. Notably, we show that residue R443, which resides in the fourth intracellular loop, also modulates AMPH‐induced DA efflux. Thus, we focus on the role of the interaction between the DAT N‐terminus and R443 in mediating the reverse transport of DA. Furthermore, we translate our molecular discoveries in vivo by using a coordinated genetic and pharmacological approach to study AMPH‐induced behaviors in Drosophila melanogasterSupport or Funding Information1F31MH114316 (JIA), AHA Predoctoral Award (AS), DA035263(AG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction between the dopamine transporter and the Gβγ subunits of the heterotrimeric G‐protein modifies transporter function and response to psychostimulants

Monoaminergic neurotransmission (dopamine, serotonin, and norepinephrine) is altered in complex psychiatric conditions such as depression, attention‐deficit hyperactivity disorder (ADHD) and drug use disorders. The dopamine transporter (DAT) clears extracellular dopamine (DA) through a sodium‐coupled transport mechanism. The fine‐tuning of DA homeostasis is maintained by multiple mechanisms involving protein‐protein interactions. We recently reported that G‐protein beta‐gamma (Gβγ) subunits bind directly to DAT. The binding of Gβγ results in a net change in the direction of the DA flux, increasing extracellular DA levels. The activation of Gβγ by a binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous systems and primary dopaminergic neurons in culture. The activation of a Gq‐subtype GPCR like the muscarinic receptor 5 (hM5R), also stimulates efflux that can be blocked with gallein, a small molecule that block the Gbg “hot spot” for binding to different effectors. A permeable TAT‐peptide containing the Gβγ interacting domain of DAT (human DAT 582 to 586) blocked the ability of mSIRK and carbachol to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. To further understand the mechanism of Gβγ regulation on DAT function, we examined whether the functional effects were linked to conformational changes in the DAT. An extracellularly‐oriented cysteine residue (C306) was used to probe conformational changes induced by the Gβγ activator peptide, mSIRK, and ligands such as cocaine, ibogaine and other modulators of DAT function. The accessibility of C306 to an impermeant thiol‐reactive reagent was significantly reduced in cells treated with mSIRK. It has previously been shown that cocaine increases the accessibility of C306 by stabilizing an outward facing conformation of the DAT. However, pretreatment of cells with mSIRK reduced the cocaine‐induced increase in C306 reactivity, suggesting that Gβγ activation limits the available binding sites for cocaine, perhaps by stabilizing an inward conformation. Finally, we wanted to test if the observed conformational changes were result of the Gβγ binding to DAT or if the binding of Gβγ is conformation‐dependent. For that we used the proximity‐ligation assay, new approach to study protein‐protein interactions, to study how the interaction DAT‐Gβγ is modified in the presence of different conformation‐specific DAT‐ligands. Collectively, our data show that Gβγ interacts directly with DAT and facilitate a shift in DAT conformation towards an inwardly‐oriented efflux mode. This novel mechanism may have important implications in the regulation of brain DA homeostasis.Support or Funding InformationNIMH IRP:ZIA MH002946‐04This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immortalized Rat Neuronal Cell Line Shows Potential as an Improved Cell Model for Dopamine Transporter Research

Dopamine (DA) is a neurotransmitter which controls several psychological and physiological processes such as reward, motivation, attention, and motor activity. Disruption of DA homeostasis is implicated in several neurological diseases such as attention‐deficit hyperactivity disorder (ADHD), schizophrenia, depression, and Parkinson disease. The DA transporter (DAT) is an integral plasma membrane protein that actively translocates extracellular DA into the presynaptic neuron. DAT serves as a primary mechanism by which synaptic DA levels and neurotransmission is spatially and temporally modulated. In addition, DAT is a major site of action for the psychostimulants cocaine and amphetamine, and for therapeutic drugs such as methylphenidate and bupropion used to treat ADHD and depression. Thus, a greater understanding of DAT functional and regulatory mechanisms is critical as this may lead to additional effective therapeutic interventions. Currently, many cell models are used in exploring DAT function and regulation. However, it is uncertain if any of these cell lines adequately replicate dopaminergic neuron physiology. Primary cultures from brain tissue appear to provide the most direct and reliable model for studying dopaminergic functions but are removed from their specialized neuronal environment and very limited in number, restricting their usefulness for many biochemical assays. In addition, primary cultures are labor intensive and challenging to prepare and maintain. To address these issues, we are examining the feasibility of the neuronal cell line, 1RB3AN27 (N27), derived from immortalized rat primary dopaminergic neurons as an improved cell model for studying DAT. We have assessed these cells for expression of key dopaminergic proteins such as tyrosine hydroxylase (TH), DOPA decarboxylase (DOPA‐DC), and native DAT via immunoblotting, finding modest TH and DOPA‐DC and very low DAT protein levels. Differentiation failed to elevate protein levels of these dopaminergic markers, although more experimental replicates are needed to confirm these observations. We are currently determining the levels of other proteins important for neuron physiology such as syntaxin 1a, vesicular monoamine transporter 2 (VMAT2), dopamine receptor D2 (D2R), and glutamate receptors (AMPA, mGluR, and NMDA). Due to very low DAT levels in both undifferentiated and differentiated cells, we transfected these cells with human (h) or rat (r) DAT, creating lines stably expressing these DAT forms. Both the rDAT and hDAT N27 lines showed robust DA uptake capacity compared to the native N27 line in which little to no DA uptake was detected, indicating no detectable DAT or norepinephrine transporter expression. These stable transformants also showed amphetamine‐stimulated DA efflux, a characteristic, pharmacological response in dopaminergic neurons. Using an ELISA specific for detecting DA, similar extracellular DA levels were detected in the rDAT N27 line compared to the parental N27 line, demonstrating DA production in these lines. To support our ELISA results, we have begun to assess N27 cells for DA production using a highly sensitive liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) assay. Taken together, our results suggest that these cells may serve as suitable neuronal models in investigating DAT function and regulation. However, further studies are required to confirm this assertion.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research Resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

3,4-Methylenedioxymethamphetamine (MDMA) Alters Synaptic Dopamine Release in the Dorsal Striatum Through Nicotinic Receptors and DAT Inhibition

An increase of extracellular dopamine (DA) has been implicated in the psychostimulant properties of 3,4-methylenedioxymethamphetamine (MDMA). Although this drug has been reported to affect the DA uptake transporter (DAT), it might activate other mechanisms to regulate the outflow of DA in the brain. Our aim was to examine the overall effects of MDMA on the release of DA in the striatum.We studied the effect of MDMA on stimulus-evoked synaptic DA release in dorsal striatal slices of mice using in vitro amperometric techniques. We also tested the effects of MDMA on the nicotine-induced responses in substantia nigra pars compacta (SNpc) neurons using intracellular electrophysiological recordings.MDMA (1–30 µM) depressed the amplitude and prolonged the decay-time of synaptic DA release in the striatum. Interestingly, in the presence of the broad nicotinic receptor antagonist mecamylamine, and the more selective α4β2 antagonist dihydroβerythroidine (DHβE), MDMA enhanced both peak and duration of DA release. A similar effect was found on cocaine-insensitive (DAT-CI) mice slices.Concentrations of MDMA higher than 100 µM enhanced striatal DA outflow that was in turn, reduced by cocaine.Electrophysiological recordings of dopaminergic neurons in SNpc showed that MDMA depressed the effects of nicotine.Our data are consistent with a prevalent MDMA-induced inhibition of the synaptic release of DA in the dorsal striatum mediated by an interaction with nicotinic receptors. This drug also blocks DAT acting on a different site from cocaine and, at higher concentrations, has amphetamine-like releasing properties.

DAT1-Genotype and Menstrual Cycle, but Not Hormonal Contraception, Modulate Reinforcement Learning: Preliminary Evidence.

Hormone by genotype interactions have been widely ignored by cognitive neuroscience. Yet, the dependence of cognitive performance on both baseline dopamine (DA) and current 17ß-estradiol (E2) level argues for their combined effect also in the context of reinforcement learning. Here, we assessed how the interaction between the natural rise of E2 in the late follicular phase (FP) and the 40 base-pair variable number tandem repeat polymorphism of the dopamine transporter (DAT1) affects reinforcement learning capacity. 30 women with a regular menstrual cycle performed a probabilistic feedback learning task twice during the early and late FP. In addition, 39 women, who took hormonal contraceptives (HC) to suppress natural ovulation, were tested during the “pill break” and the intake phase of HC. The present data show that DAT1-genotype may interact with transient hormonal state, but only in women with a natural menstrual cycle. We found that carriers of the 9-repeat allele (9RP) experienced a significant decrease in the ability to avoid punishment from early to late FP. Neither homozygote subjects of the 10RP allele, nor subjects from the HC group showed a change in behavior between phases. These data are consistent with neurobiological studies that found that rising E2 may reverse DA transporter function and could enhance DA efflux, which would in turn reduce punishment sensitivity particularly in subjects with a higher transporter density to begin with. Taken together, the present results, although based on a small sample, add to the growing understanding of the complex interplay between different physiological modulators of dopaminergic transmission. They may not only point out the necessity to control for hormonal state in behavioral genetic research, but may offer new starting points for studies in clinical settings.

CaMKII inhibition ameliorated levodopa-induced dyskinesia by downregulating tyrosine hydroxylase activity in an experimental model of Parkinson’s disease

Levodopa (L-dopa) remains the best treatment for Parkinson’s disease (PD). However, long-term L-dopa treatment induces dyskinesia. The mechanism of L-dopa-induced dyskinesia (LID) is not fully understood. Enhanced activity of protein kinase A (PKA) and pulsatile dopamine (DA) stimulation plays an important role in LID. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for DA synthesis. Decreased TH activity causes reduced pulsatile DA stimulation, which in turn reduces LID. Moreover, TH is a substrate of CaMKII. However, it is unknown whether inhibition of CaMKII reduces LID by downregulating the activity of TH. In this study, we found that CaMKII antagonist KN-93 reduced DA released in PC12 cells; in the meantime, KN-93 reduced phosphorylated levels of CaMKIIα and TH at Ser 40. Intrastriatal administration of KN-93 reduced LID without affecting the antiparkinsonian effect of L-dopa in PD mice. Mechanistically, KN-93 treatmentreduced phosphorylated CaMKIIα levels and subsequently downregulated phosphorylated TH at Ser 40 expression. Consequently, extracellular DA efflux was reduced andthe activation threshold of the PKA pathway was lowered. Moreover, KN-93 treatment reduced the expression of Arc and Penk, two immediate early genes, induced by chronic L-dopa. These data indicate that inhibition of CaMKIIα decreases LID at least partially by suppressing TH activity and subsequently reducing extracellular DA efflux and the activity of the PKA pathway, suggesting that CaMKIIα may be an alternative target for the treatment of LID.

Striatal norepinephrine efflux in l-DOPA-induced dyskinesia

l-DOPA remains the primary treatment for Parkinson's disease (PD). Unfortunately, its therapeutic benefits are compromised by the development of abnormal involuntary movements (AIMs) known as l-DOPA-induced dyskinesia (LID). The norepinephrine (NE) system originating in the locus coeruleus is profoundly affected in PD and known to influence dopamine (DA) signaling. However, the effect of noradrenergic loss on l-DOPA-induced striatal monoamine efflux and Parkinsonian motor behavior remains controversial and is frequently overlooked in traditional animal models of LID. Thus, the current study sought to determine whether degeneration of the DA and/or NE system(s) altered l-DOPA-induced striatal monoamine efflux in hemiparkinsonian rats with additional NE loss induced by the potent NE-toxin α DA beta hydroxylase (DBH)-saporin. Sham-, DA-, NE-, and dual DA + NE-lesioned rats were treated with l-DOPA (6 mg/kg, s.c.) for 2 weeks. Thereafter, l-DOPA-mediated striatal monoamine efflux was measured with in vivo microdialysis, and concurrent AIMs testing occurred to determine responsiveness to l-DOPA. Noradrenergic lesions exacerbated parkinsonian motor deficits but did not significantly alter LID expression or corresponding l-DOPA-induced striatal monoamine efflux. Interestingly, l-DOPA-induced striatal NE efflux rather than DA efflux, corresponded more closely with dyskinesia severity. Moreover, marked reductions in striatal NE tissue concentration did not appear to impact l-DOPA-induced striatal NE efflux. The current study implicates l-DOPA-induced striatal NE as an important factor in LID expression and demonstrates the importance of developing treatment strategies that co-modulate the NE and DA systems.

Norepinephrine in the Medial Pre-frontal Cortex Supports Accumbens Shell Responses to a Novel Palatable Food in Food-Restricted Mice Only.

Previous findings from this laboratory demonstrate: (1) that different classes of addictive drugs require intact norepinephrine (NE) transmission in the medial pre Frontal Cortex (mpFC) to promote conditioned place preference and to increase dopamine (DA) tone in the nucleus accumbens shell (NAc Shell); (2) that only food-restricted mice require intact NE transmission in the mpFC to develop conditioned preference for a context associated with milk chocolate; and (3) that food-restricted mice show a significantly larger increase of mpFC NE outflow then free fed mice when experiencing the palatable food for the first time. In the present study we tested the hypothesis that only the high levels of frontal cortical NE elicited by the natural reward in food restricted mice stimulate mesoaccumbens DA transmission. To this aim we investigated the ability of a first experience with milk chocolate to increase DA outflow in the accumbens Shell and c-fos expression in striatal and limbic areas of food–restricted and ad-libitum fed mice. Moreover, we tested the effects of a selective depletion of frontal cortical NE on both responses in either feeding group. Only in food-restricted mice milk chocolate induced an increase of DA outflow beyond baseline in the accumbens Shell and a c-fos expression larger than that promoted by a novel inedible object in the nucleus accumbens. Moreover, depletion of frontal cortical NE selectively prevented both the increase of DA outflow and the large expression of c-fos promoted by milk chocolate in the NAc Shell of food-restricted mice. These findings support the conclusion that in food-restricted mice a novel palatable food activates the motivational circuit engaged by addictive drugs and support the development of noradrenergic pharmacology of motivational disturbances.

Exploring the consequences of social defeat stress and intermittent ethanol drinking on dopamine dynamics in the rat nucleus accumbens

The current study aimed to explore how presynaptic dopamine (DA) function is altered following brief stress episodes and chronic ethanol self-administration and whether these neuroadaptations modify the acute effects of ethanol on DA dynamics. We used fast-scan cyclic voltammetry to evaluate changes in DA release and uptake parameters in rat nucleus accumbens brain slices by analyzing DA transients evoked through single pulse electrical stimulation. Adult male rats were divided into four groups: ethanol-naïve or ethanol drinking (six week intermittent two-bottle choice) and stressed (mild social defeat) or nonstressed. Results revealed that the mild stress significantly increased DA release and uptake in ethanol-naïve subjects, compared to nonstressed controls. Chronic ethanol self-administration increased the DA uptake rate and occluded the effects of stress on DA release dynamics. Bath-applied ethanol decreased stimulated DA efflux in a concentration-dependent manner in all groups; however, the magnitude of this effect was blunted by either stress or chronic ethanol, or by a combination of both procedures. Together, these findings suggest that stress and ethanol drinking may promote similar adaptive changes in accumbal presynaptic DA release measures and that these changes may contribute to the escalation in ethanol intake that occurs during the development of alcohol use disorder.

Analysis of the roles of μ receptor subtypes in the regulation of acetylcholine and dopamine efflux in the nucleus accumbens of freely moving rats

Analysis of the roles of μ receptor subtypes in the regulation of acetylcholine and dopamine efflux in the nucleus accumbens of freely moving rats

Activation of accumbal GABA<sub>B</sub> receptors inhibits δ<sub>1</sub> and δ<sub>2</sub> receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats

Activation of accumbal GABA<sub>B</sub> receptors inhibits δ<sub>1</sub> and δ<sub>2</sub> receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats

Functional properties of dopamine transporter oligomers after copper linking.

Although it is universally accepted that dopamine transporters (DATs) exist in monomers, dimers and tetramers (i.e. dimers of dimers), it is not known whether the oligomeric organization of DAT is a prerequisite for its ability to take up dopamine (DA), or whether each DAT protomer, the subunit of quaternary structure, functions independently in terms of DA translocation. In this study, copper phenanthroline (CuP) was used to selectively target surface DAT: increasing concentrations of CuP gradually cross-linked natural DAT dimers in LLC-PK1 cells stably expressing hDAT and thereby reduced DA uptake functionality until all surface DATs were inactivated. DATs that were not cross-linked by CuP showed normal DA uptake with DA Km at ~ 0.5μM and DA efflux with basal and amphetamine-induced DA efflux as much as control values. The cocaine analog 2β-carbomethoxy-3β-[4-fluorophenyl]-tropane (CFT) was capable to bind to copper-cross-linked DATs, albeit with an affinity more than fivefold decreased (Kd of CFT=109nM after cross-linking vs 19nM before). A kinetic analysis is offered describing the changing amounts of dimers and monomers with increasing [CuP], allowing the estimation of dimer functional activity compared with a DAT monomer. Consonant with previous conclusions for serotonin transporter and NET that only one protomer of an oligomer is active at the time, the present data indicated a functional activity of the DAT dimer of 0.74 relative to a monomer.

The sigma-1 receptor modulates methamphetamine dysregulation of dopamine neurotransmission

Dopamine neurotransmission is highly dysregulated by the psychostimulant methamphetamine, a substrate for the dopamine transporter (DAT). Through interactions with DAT, methamphetamine increases extracellular dopamine levels in the brain, leading to its rewarding and addictive properties. Methamphetamine also interacts with the sigma-1 receptor (σ1R), an inter-organelle signaling modulator. Using complementary strategies, we identified a novel mechanism for σ1R regulation of dopamine neurotransmission in response to methamphetamine. We found that σ1R activation prevents methamphetamine-induced, DAT-mediated increases in firing activity of dopamine neurons. In vitro and in vivo amperometric measurements revealed that σ1R activation decreases methamphetamine-stimulated dopamine efflux without affecting basal dopamine neurotransmission. Consistent with these findings, σ1R activation decreases methamphetamine-induced locomotion, motivated behavior, and enhancement of brain reward function. Notably, we revealed that the σ1R interacts with DAT at or near the plasma membrane and decreases methamphetamine-induced Ca2+ signaling, providing potential mechanisms. Broadly, these data provide evidence for σ1R regulation of dopamine neurotransmission and support the σ1R as a putative target for the treatment of methamphetamine addiction.

Dopamine transporter phosphorylation site threonine 53 is stimulated by amphetamines and regulates dopamine transport, efflux, and cocaine analog binding

The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr-53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr-53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+ Together these findings support a role for Thr-53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.

Phosphorylation of the Amino Terminus of the Dopamine Transporter: Regulatory Mechanisms and Implications for Amphetamine Action.

Amphetamines (AMPHs) are potent psychostimulants that are widely used and abused, with profound medical and societal impact. Their actions at dopaminergic neurons are thought to mediate their therapeutic efficacy as well as their liability for abuse and dependence. AMPHs target the dopamine transporter (DAT), the plasmalemmal membrane protein that mediates the inactivation of released dopamine (DA) through its reuptake. AMPHs act as substrates for DAT and are known to cause mobilization of dopamine (DA) to the cell exterior via DAT-mediated reverse transport (efflux). It has become increasingly evident that the mechanisms that regulate AMPH-induced DA efflux are distinct from those that regulate DA uptake. Central to these mechanisms is the phosphorylation of the DAT amino (N)-terminus, which has been repeatedly demonstrated to facilitate DAT-mediated DA efflux, without impacting other aspects of DAT physiology. This review aims to summarize the current status of knowledge regarding DAT N-terminal phosphorylation and its regulation by protein modulators and the membrane microenvironment. A better understanding of these mechanisms may lead to the identification of novel therapeutic approaches that interfere selectively with the pharmacological effects of AMPHs without altering the physiological function of DAT.

Stimulation of accumbal GABAA receptors inhibits delta2-, but not delta1-, opioid receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats

The nucleus accumbens contains delta-opioid receptors that may reduce inhibitory neurotransmission. Reduction in GABAA receptor-mediated inhibition of accumbal dopamine release due to delta-opioid receptor activation should be suppressed by stimulating accumbal GABAA receptors. As delta-opioid receptors are divided into delta2- and delta1-opioid receptors, we analysed the effects of the GABAA receptor agonist muscimol on delta2- and delta1-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were administered intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 25–50min infusions. The delta2-opioid receptor agonist deltorphin II (25.0nmol)- and delta1-opioid receptor agonist DPDPE (5.0nmol)-induced increases in dopamine efflux were inhibited by the delta2-opioid receptor antagonist naltriben (1.5nmol) and the delta1-opioid receptor antagonist BNTX (150.0pmol), respectively. Muscimol (250.0pmol) inhibited deltorphin II (25.0nmol)-induced dopamine efflux. The GABAA receptor antagonist bicuculline (50.0pmol), which failed to affect deltorphin II (25.0nmol)-induced dopamine efflux, counteracted the inhibitory effect of muscimol on deltorphin II-induced dopamine efflux. Neither muscimol (250.0pmol) nor bicuculline (50.0 and 500.0pmol) altered DPDPE (5.0nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABAA receptor-mediated inhibition of dopaminergic activity is necessary to produce delta2-opioid receptor-induced increase in accumbal dopamine efflux. This study indicates that activation of delta2- but not delta1-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABAA receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux.