A role for cortical dopamine in the paradoxical calming effects of psychostimulants.

Psychostimulants have a paradoxical calming effect in the treatment of attention deficit hyperactivity disorder (ADHD), but their mechanism of action is unclear. Studies using dopamine (DA) transporter (DAT) knockout (KO) mice have suggested that the paradoxical calming effect of psychostimulants might occur through actions on serotonin (5-HT) neurotransmission. However, newer non-stimulant drugs, such as atomoxetine and guanfacine, suggest that targeting the norepinephrine (NE) system in the prefrontal cortex (PFC) might explain this paradoxical calming effect. Thus, we sought to clarify the mechanism of this paradoxical action of psychostimulants. Our ex vivo efflux experiments reveal that the NE transporter (NET) blocker desipramine elevates both norepinephrine (NE) and dopamine (DA), but not 5-HT levels, in PFC tissue slices from wild-type (WT) and DAT-KO, but not NET-KO mice. However, the 5-HT transporter (SERT) inhibitor fluoxetine elevates only 5-HT in all three genotypes. Systemic administration of desipramine or fluoxetine inhibits hyperactivity in DAT-KO mice, whereas local PFC infusion of desipramine alone produced this same effect. In contrast, pharmacological NE depletion and DA elevation using nepicastat also inhibits hyperactivity in DAT-KO mice. Together, these data suggest elevation of PFC DA and not NE or 5-HT, as a convergent mechanism for the paradoxical effects of psychostimulants observed in ADHD therapy.

Effect of Urate-Elevating Inosine on Early Parkinson Disease Progression: The SURE-PD3 Randomized Clinical Trial.

Importance: Urate elevation, despite associations with crystallopathic, cardiovascular, and metabolic disorders, has been pursued as a potential disease-modifying strategy for Parkinson disease (PD) based on convergent biological, epidemiological, and clinical data. Objective: To determine whether sustained urate-elevating treatment with the urate precursor inosine slows early PD progression. Design, Participants, and Setting: Randomized, double-blind, placebo-controlled, phase 3 trial of oral inosine treatment in early PD. A total of 587 individuals consented, and 298 with PD not yet requiring dopaminergic medication, striatal dopamine transporter deficiency, and serum urate below the population median concentration (<5.8 mg/dL) were randomized between August 2016 and December 2017 at 58 US sites, and were followed up through June 2019. Interventions: Inosine, dosed by blinded titration to increase serum urate concentrations to 7.1-8.0 mg/dL (n = 149) or matching placebo (n = 149) for up to 2 years. Main Outcomes and Measures: The primary outcome was rate of change in the Movement Disorder Society Unified Parkinson Disease Rating Scale (MDS-UPDRS; parts I-III) total score (range, 0-236; higher scores indicate greater disability; minimum clinically important difference of 6.3 points) prior to dopaminergic drug therapy initiation. Secondary outcomes included serum urate to measure target engagement, adverse events to measure safety, and 29 efficacy measures of disability, quality of life, cognition, mood, autonomic function, and striatal dopamine transporter binding as a biomarker of neuronal integrity. Results: Based on a prespecified interim futility analysis, the study closed early, with 273 (92%) of the randomized participants (49% women; mean age, 63 years) completing the study. Clinical progression rates were not significantly different between participants randomized to inosine (MDS-UPDRS score, 11.1 [95% CI, 9.7-12.6] points per year) and placebo (MDS-UPDRS score, 9.9 [95% CI, 8.4-11.3] points per year; difference, 1.26 [95% CI, -0.59 to 3.11] points per year; P = .18). Sustained elevation of serum urate by 2.03 mg/dL (from a baseline level of 4.6 mg/dL; 44% increase) occurred in the inosine group vs a 0.01-mg/dL change in serum urate in the placebo group (difference, 2.02 mg/dL [95% CI, 1.85-2.19 mg/dL]; P<.001). There were no significant differences for secondary efficacy outcomes including dopamine transporter binding loss. Participants randomized to inosine, compared with placebo, experienced fewer serious adverse events (7.4 vs 13.1 per 100 patient-years) but more kidney stones (7.0 vs 1.4 stones per 100 patient-years). Conclusions and Relevance: Among patients recently diagnosed as having PD, treatment with inosine, compared with placebo, did not result in a significant difference in the rate of clinical disease progression. The findings do not support the use of inosine as a treatment for early PD. Trial Registration: ClinicalTrials.gov Identifier: NCT02642393.

Psychomotor impairments and therapeutic implications revealed by a mutation associated with infantile Parkinsonism-Dystonia.

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.

Chronic antipsychotic treatment exerts limited effects on the mania-like behavior of dopamine transporter knockdown mice.

BACKGROUND: Bipolar disorder is a life-threatening disorder linked to dopamine transporter (DAT) polymorphisms, with reduced DAT levels seen in positron emission tomography and postmortem brains. AIMS: The purpose of this study was to examine the effects of approved antipsychotics on DAT dysfunction-mediated mania behavior in mice. METHODS: DAT knockdown mice received either D2-family receptor antagonist risperidone or asenapine and mania-related behaviors were assessed in the clinically-relevant behavioral pattern monitor to assess spontaneous exploration. RESULTS: Chronic risperidone did not reverse mania-like behavior in DAT knockdown mice. Chronic asenapine reduced mania behavior but this effect was more pronounced in wild-type littermates than in DAT knockdown mice. CONCLUSION: Taken together, these findings suggest that while acute antipsychotic treatment may be beneficial in management of bipolar mania, more targeted therapeutics may be necessary for long-term treatment. Specific investigation into DAT-targeting drugs could improve future treatment of bipolar mania.

Enhanced Dopamine Transmission and Hyperactivity in the Dopamine Transporter Heterozygous Mice Lacking the D3 Dopamine Receptor.

Dopamine transporter knockout (DATk) mice are known to demonstrate profound hyperactivity concurrent with elevated (5-fold) extracellular dopamine in the basal ganglia. At the same time, heterozygous DAT mice (DATh) demonstrate a 2-fold increase in dopamine levels yet only a marginal elevation in locomotor activity level. Another model of dopaminergic hyperactivity is the D3 dopamine receptor knockout (D3k) mice, which present only a modest hyperactivity phenotype, predominately manifested as stereotypical behaviors. In the D3k mice, the hyperactivity is also correlated with elevated extracellular dopamine levels (2-fold) in the basal ganglia. Cross-breeding was used to evaluate the functional consequences of the deletion of both genes. In the heterozygous DAT mice, inactivation of the D3R gene (DATh/D3k) resulted in significant hyperactivity and further elevation of striatal extracellular dopamine above levels observed in respective single mutant mice. The decreased weight of DATk mice was evident regardless of the D3 dopamine receptor genotype. In contrast, measures of thermoregulation revealed that the marked hypothermia of DATk mice (-2 °C) was reversed in double knockout mice. Thus, the extracellular dopamine levels elevated by prolonging uptake could be elevated even further by eliminating the D3 receptor. These data also suggest that the hypothermia observed in DATk mice may be mediated through D3 receptors.

[Influence of the DAT gene knockout on exchange of essential and toxic trace elements in rats].

The maintenance of energy homeostasis of the body according to modern data is carried out with the active participation of dopaminergic neurons of the central nervous system. The synthesis and metabolism of dopamine (DA) occurs both in the brain and in peripheral tissues. Violation of the synthesis and metabolism of DA is considered as a link in the vicious cycle which it formed during the development of diet-induced obesity. According to modern data, a number of essential and toxic trace elements, such as Cd, Al, As, Mn, Fe, Cu, Zn, are actively involved in the exchange of DA in the brain and peripheral organs and tissues. One way to assess this relationship is to compare changes in the microelement status of the organism when consuming hypercaloric diets in animals with normal and impaired DA transport. The latter can be animals with a knockout of the DAT transporter gene, which performs DA reabsorption with subsequent storage in the composition of secretory granules. The aim is a comparative study of the content of a number of essential and toxic elements in the brain, liver, and kidneys of rats that differ in the allelic variants of the DAT gene fed balanced diet and the diet with an excess of energy value. Material and methods. The study was carried out on 30 male rats of the DAT-KO knockout line (homozygotes DAT-/- and heterozygotes DAT+/-), 8-10 weeks old, and 13 males rats of the outbred Wistar line (DAT+/+) of the same age. For 62 days the animals (6 groups) received a semi-synthetic diet containing essential elements in the salt mixture or a similar high-fat-highcarbohydrate diet (HFCD) with 30% fat and 20% fructose solution instead of drinking water. The content of 16 trace elements (Fe, Mg, Cu, Mn, Co, Se, Zn, Cr, V, Cs, Ag, Al, Cd, As, Pb, Ni) were determined by inductively coupled plasma mass spectrometry in the liver, kidneys, and brain of rats with a knockout of the dopamine DAT transporter gene: homozygotes (DAT -/-) and heterozygotes (DAT+/-), as well as wild-type rats (DAT+/+) of the Wistar strain. Results and discussion. In the liver, DAT knockout led to an increase in the content of As, Cd, Co, and Cs and a decrease in Fe; in the kidneys - to an increase in the levels of Pb, As, Cd and Se, in the brain - an increase in the content of most of the studied trace elements, including Pb, As, Cs, Al and Cu. Conclusion. Against the background of consumption of HFCD, the effect of DAT knockout on the content of a number of elements was more pronounced compared with the consumption of the control diet. The revealed changes in the trace element content in DAT knockout rats are considered in terms of the effect of DA metabolism in the central nervous system and in peripheral tissues on the status of trace elements.

Rats Lacking Dopamine Transporter Display Increased Vulnerability and Aberrant Autonomic Response to Acute Stress.

The activity of the hypothalamus-pituitary-adrenal (HPA) axis is pivotal in homeostasis and presides the adaptative response to stress. Dopamine Transporter (DAT) plays a key role in the regulation of the HPA axis. We used young adult female DAT Knockout (KO) rats to assess the effects of DAT ablation (partial, heterozygous DAT+/-, or total, homozygous DAT-/-) on vulnerability to stress. DAT-/- rats show profound dysregulation of pituitary homeostasis, in the presence of elevated peripheral corticosterone, before and after acute restraint stress. During stress, DAT-/- rats show abnormal autonomic response at either respiratory and cardiovascular level, and delayed body temperature increase. DAT+/- rats display minor changes of hypophyseal homeostatic mechanisms. These rats display a similar pituitary activation to that of the control animals, albeit in the presence of higher release of peripheral corticosterone than DAT-/- after stress, and reduced temperature during stress. Our data indicate that DAT regulates the HPA axis at both the central and peripheral level, including autonomic function during stress. In particular, the partial deletion of DAT results in increased vulnerability to stress in female rats, which display central and peripheral alterations that are reminiscent of PTSD, and they might provide new insights in the pathophysiology of this disorder.

Deficit in working memory and abnormal behavioral tactics in dopamine transporter knockout rats during training in the 8-arm maze.

Understanding the role of the dopamine system in learning and memory processes is very important for uncovering central mechanisms underlying complex behavioral responses that can be impaired in patients with neuropsychiatric disorders caused by dopamine system dysfunction. One of the most useful animal models for dopaminergic dysregulation is the strain of dopamine transporter knockout (DAT-KO) rats that have no dopamine re-uptake and thus elevated extracellular dopamine levels. It is known that dopamine is involved in various cognitive processes such as learning, memory and attention. This investigation was focused on the ability of DAT-KO rats to learn and perform a behavioral task in the 8-arm radial maze test. It was found that DAT-KO rats are able to learn the behavioral task, but the level of task performance did not reach that of WT group. The behavioral tactics used by animals during training significantly differ in mutants. The behavioral tactics used by DAT-KO rats involved perseverations and resulted in worse task fulfillment in comparison to wild-type controls. The data obtained indicate that deficient dopamine reuptake results in an impairment of working memory and perseverative behavioral tactics in DAT-KO rats.

Correlations between dopaminergic dysfunction and abnormal metabolic network activity in REM sleep behavior disorder.

Striatal dopamine transporter (DAT) deficiency and abnormal expression of Parkinson's disease (PD)-related pattern (PDRP) have been observed in patients with idiopathic REM sleep behavior disorder (IRBD). This study aimed to investigate the correlations between these two measures with comparison to PD using a dual tracer imaging design. Age-matched 37 IRBD patients, 86 PD patients, and 15 control subjects underwent concurrent PET scans with 11C-CFT to quantify dopaminergic dysfunction and 18F-FDG to quantify PDRP expression. IRBD patients were divided into two subgroups: those with relatively normal (IRBD-RN) or abnormal (IRBD-AB) striatal DAT binding. Significantly decreased DAT binding and increased PDRP scores were present in all patient groups, except for IRBD-RN, relative to the controls. There was a significant effect of hemisphere and hemisphere × group interaction for DAT binding but not for PDRP expression. Significant correlations were observed between DAT binding and PDRP expression in the IRBD-AB and PD groups but not in the IRBD-RN group. IRBD patients present with an intermediate state in striatal DAT distribution and PDRP activity between PD and normal controls. The modest correlations between the two measures in both IRBD and PD suggest that differences in network activity cannot be fully explained by nigrostriatal dopaminergic denervation.

Discovery of ß-Arrestin Biased, Orally Bioavailable, and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators.

Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders, but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.

Individual parkinsonian motor signs and striatal dopamine transporter deficiency: a study with [I-123]FP-CIT SPECT.

INTRODUCTION: Total parkinsonian motor symptom severity correlates with presynaptic striatal dopamine function in patients with Parkinson's disease. There is a lack of studies that have investigated the associations between parkinsonian motor signs and striatal dopaminergic deficiency in patients with parkinsonism of an unknown origin. Identification of specific motor signs associated with the highest likelihood of striatal dopamine deficiency could aid the differential diagnostics of parkinsonian and tremor syndromes. METHODS: In this cross-sectional clinical and imaging study, detailed motor examinations were performed for 221 patients with parkinsonism or tremor of an unknown origin immediately before dopamine transporter (DAT) [I-123]FP-CIT SPECT imaging. Region-of-interest and voxel-based methods were used to investigate striatal DAT deficiency in relation to individual motor signs. RESULTS: Upper extremity rigidity and facial expression were the only motor signs that differentiated patients with normal and abnormal striatal DAT function. The presence of any upper extremity rigidity showed the highest likelihood of DAT deficiency (OR 4.79, 95% CI 1.56-14.75, P = 0.006) followed by reduced facial expression (OR 2.14, 95% CI 1.14-4.00, P = 0.018). In patients with DAT deficits, reduced facial expression was associated with DAT deficiency specifically in the caudate nucleus, and increased upper extremity rigidity was associated with DAT loss in the dorsal putamen (FWE-corrected P < 0.05). CONCLUSIONS: Increased upper extremity muscle tone and hypomimia are independently associated with a higher likelihood of striatal hypodopaminergic imaging finding. This information can be used as a factor when the clinical need of auxiliary investigations, such as DAT SPECT, is considered for patients with parkinsonism.

Behavioral characterization of DAT-KO rats and evidence of asocial-like phenotypes in DAT-HET rats: The potential involvement of norepinephrine system.

Dopamine (DA) is a key neurotransmitter of the central nervous system, whose availability is regulated by the dopamine transporter (DAT). Deletion of DAT gene leading to hyperdopaminergia was previously performed on mouse models. This enabled recapitulation of the core symptoms of Attention-Deficit / Hyper-activity Disorder (ADHD), which include hyperactivity, inattention and cognitive impairment. We used recently developed DAT knockout (DAT-KO) rats to carry out further behavioral profiling on this novel model of hyperdopaminergia. DAT-KO rats display elevated locomotor activity and restless environmental exploration, associated with a transient anxiety profile. Furthermore, these rats show pronounced stereotypy and compulsive-like behavior at the Marble-Burying test. Homozygous DAT-KO rats mantain intact social interaction when tested in a social-preference task, while heterozygous (HET) rats show high inactivity associated with close proximity to the social stimulus. Ex-vivo evaluation of brain catecholamines highlighted increased levels of norepinephrine in the hippocampus and hypothalamus exclusively of heterozygous rats. Taken together, our data present evidence of unexpected asocial tendencies in heterozygous (DAT-HET) rats associated with neurochemical alterations in norepinephrine neurotransmission. We shed light on the behavioral and neurochemical consequences of altered DAT function in a higher, more complex model of hyperdopaminergia. Unraveling the role of DA neurotransmission in DAT-KO rats has very important implications in the understanding of many psychiatric illnesses, including ADHD, where alterations in DA system have been demonstrated.

No effect of sex on ethanol intake and preference after dopamine transporter (DAT) knockdown in adult mice.

RATIONALE: Dopamine levels are controlled in part by transport across the cell membrane by the dopamine transporter (DAT), and recent evidence showed that a polymorphism in the gene encoding DAT is associated with alcoholism. However, research in animal models using DAT knockout mice has yielded conflicting results. OBJECTIVES: The present study was planned to evaluate the effects of DAT knockdown in the nucleus accumbens (Nacc) on voluntary ethanol consumption and preference in male and female C57BL/6J mice. METHODS: For this purpose, animals were stereotaxically injected with DAT siRNA-expressing lentiviral vectors in the Nacc, and using a voluntary, continuous access two-bottle choice model of alcohol, we investigated the importance of accumbal DAT expression in voluntary alcohol intake and preference. We also investigated the effects of DAT knockdown on saccharin and quinine consumption and ethanol metabolism. RESULTS: We show that females consumed more alcohol than males. Interestingly, DAT knockdown in the Nacc significantly decreased alcohol intake and preference in both groups, but no significant sex by group interaction was observed. Also, DAT knockdown did not alter total fluid consumption, saccharin or quinine consumption, or blood ethanol concentrations. Using Pearson correlation, results indicated a strong positive relationship between DAT mRNA expression and ethanol consumption and preference. CONCLUSIONS: Taken together, these data provide further evidence that DAT plays an important role in controlling ethanol intake and that accumbal DAT contributes in the modulation of the reinforcing effects of ethanol. Overall, the results suggest that DAT inhibitors may be valuable in the pharmacotherapy of alcoholism.

Dopamine transporter (DAT) knockdown in the nucleus accumbens improves anxiety- and depression-related behaviors in adult mice.

Many epidemiological and clinical studies have demonstrated a strong comorbidity between anxiety and depression, and a number of experimental studies indicates that the dopamine transporter (DAT) is involved in the pathophysiology of anxiety and depression. However, studies using laboratory animals have yielded inconclusive results. The aim of the present study was to examine the effects of DAT manipulation on anxiety- and depression-like behaviors in mice. For this purpose, animals were stereotaxically injected with DAT siRNA-expressing lentiviral vectors (siDAT) in the caudate putamen (CPu) or in the nucleus accumbens (Nacc) and the behavioral outcomes were assessed using the open-field (OF), elevated-plus maze (EPM), light-dark box (LDB), sucrose preference (SPT), novelty suppressed feeding (NSF), and forced-swim (FST) tests. The results showed that in the Nacc, but not in the CPu, siDAT increased the time spent at the center of the arena and decreased the number of fecal boli in the OF test. In the EPM and LDB tests, Nacc siDAT injection increased the entries and time spent on open arms, and increased the time spent in the light side of the box, respectively, suggesting an anxiolytic-like activity. In addition, siDAT, in the Nacc, induced significant antidepressant-like effects, evidenced by increased sucrose preference, shorter latency to feed in the NSF test, and decreased immobility time in the FST. Most importantly, Pearson's test clearly showed significant correlations between DAT mRNA in the Nacc with anxiety and depression parameters. Overall, these results suggest that low DAT levels, in the Nacc, might act as protective factors against anxiety and depression. Therefore, targeting DAT activity might be a very attractive approach to tackle affective disorders.

Reward-enhancing effect of methylphenidate is abolished in dopamine transporter knockout mice: A model of attention-deficit/hyperactivity disorder.

AIM: Attention-deficit/hyperactivity disorder is a heterogeneous neurobiological disorder that is characterized by inattention, impulsivity, and an increase in motor activity. Although methylphenidate has been used as a medication for decades, unknown is whether methylphenidate treatment can cause drug dependence in patients with attention-deficit/hyperactivity disorder. This study investigated the reward-enhancing effects of methylphenidate using intracranial self-stimulation in an animal model of attention-deficit/hyperactivity disorder, dopamine transporter knockout mice. METHODS: For the intracranial self-stimulation procedures, the mice were trained to nosepoke to receive direct electrical stimulation via an electrode that was implanted in the lateral hypothalamus. After the acquisition of nosepoke responding for intracranial self-stimulation, the effects of methylphenidate on intracranial self-stimulation were investigated. RESULTS: In the progressive-ratio procedure, dopamine transporter knockout mice exhibited an increase in intracranial self-stimulation compared with wild-type mice. Treatment with 5 and 10 mg/kg methylphenidate increased intracranial self-stimulation responding in wild-type mice. Methylphenidate at the same doses did not affect intracranial self-stimulation responding in dopamine transporter knockout mice. We then investigated the effects of high-dose methylphenidate (60 mg/kg) in a rate-frequency procedure. High-dose methylphenidate significantly decreased intracranial self-stimulation responding in both wild-type and dopamine transporter knockout mice. CONCLUSIONS: These results suggest that low-dose methylphenidate alters the reward system (ie, increases intracranial self-stimulation responding) in wild-type mice via dopamine transporter inhibition, whereas dopamine transporter knockout mice do not exhibit such alterations. High-dose methylphenidate appears to suppress intracranial self-stimulation responding not through dopamine transporter inhibition but rather through other mechanisms. These results support the possibility that methylphenidate treatment for attention-deficit/hyperactivity disorder does not increase the risk of drug dependence, in attention-deficit/hyperactivity disorder patients with dopamine transporter dysfunction.

Mice with reduced DAT levels recreate seasonal-induced switching between states in bipolar disorder.

Developing novel therapeutics for bipolar disorder (BD) has been hampered by limited mechanistic knowledge how sufferers switch between mania and depression-how the same brain can switch between extreme states-described as the "holy grail" of BD research. Strong evidence implicates seasonally-induced switching between states, with mania associated with summer-onset, depression with winter-onset. Determining mechanisms of and sensitivity to such switching is required. C57BL/6J and dopamine transporter hypomorphic (DAT-HY 50% expression) mice performed a battery of psychiatry-relevant behavioral tasks following 2-week housing in chambers under seasonally relevant photoperiod extremes. Summer-like and winter-like photoperiod exposure induced mania-relevant and depression-relevant behaviors respectively in mice. This behavioral switch paralleled neurotransmitter switching from dopamine to somatostatin in hypothalamic neurons (receiving direct input from the photoperiod-processing center, the suprachiasmatic nucleus). Mice with reduced DAT expression exhibited hypersensitivity to these summer-like and winter-like photoperiods, including more extreme mania-relevant (including reward sensitivity during reinforcement learning), and depression-relevant (including punishment-sensitivity and loss-sensitivity during reinforcement learning) behaviors. DAT mRNA levels switched in wildtype littermate mice across photoperiods, an effect not replicated in DAT hypomorphic mice. This inability to adjust DAT levels to match photoperiod-induced neurotransmitter switching as a homeostatic control likely contributes to the susceptibility of DAT hypormophic mice to these switching photoperiods. These data reveal the potential contribution of photoperiod-induced neuroplasticity within an identified circuit of the hypothalamus, linked with reduced DAT function, underlying switching between states in BD. Further investigations of the circuit will likely identify novel therapeutic targets to block switching between states.

Dopamine D3 receptor blockade rescues hyper-dopamine activity-induced deficit in novel object recognition memory.

Patients afflicted with bipolar disorder demonstrate significant impairments in recognition and episodic memory during acute depressive and manic episodes. These impairments and the related pathophysiology may result from over-activation of the brain dopamine (DA) system. In order to model overactive DA transmission in a well-established novel object recognition (NOR) memory test, we used DA transporter knockdown (DAT-KD) mice, which exhibit reduced DAT expression and display hyper-dopaminergic phenotypes. DAT-KD mice exhibited impaired NOR memory compared to wild-type (WT) mice. This impairment was prevented by administration of FAUC365, a DA D3 receptor (D3R) selective antagonist, prior to object learning. Similarly, D3R knockout (KO)/DAT-KD double mutant mice displayed performance in the NOR test that was comparable to WT mice, suggesting that deficiencies in NOR performance in DAT-KD mice can be compensated by diminishing D3R signaling. GBR12909, a DAT blocker, also impaired NOR performance in WT mice, but not in D3R KO mice. Impaired NOR performance in GBR12909-treated WT mice was also prevented by pretreatment with FAUC365. Together, these findings indicate that reduced DAT activity can impair recognition memory in the NOR test, and D3R appears to be necessary to mediate this effect.

Pronounced Hyperactivity, Cognitive Dysfunctions, and BDNF Dysregulation in Dopamine Transporter Knock-out Rats.

Dopamine (DA) controls many vital physiological functions and is critically involved in several neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. The major function of the plasma membrane dopamine transporter (DAT) is the rapid uptake of released DA into presynaptic nerve terminals leading to control of both the extracellular levels of DA and the intracellular stores of DA. Here, we present a newly developed strain of rats in which the gene encoding DAT knockout Rats (DAT-KO) has been disrupted by using zinc finger nuclease technology. Male and female DAT-KO rats develop normally but weigh less than heterozygote and wild-type rats and demonstrate pronounced spontaneous locomotor hyperactivity. While striatal extracellular DA lifetime and concentrations are significantly increased, the total tissue content of DA is markedly decreased demonstrating the key role of DAT in the control of DA neurotransmission. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, the partial Trace Amine-Associated Receptor 1 (TAAR1) agonist RO5203648 ((S)-4-(3,4-Dichloro-phenyl)-4,5-dihydro-oxazol-2-ylamine) and haloperidol. DAT-KO rats also demonstrate a deficit in working memory and sensorimotor gating tests, less propensity to develop obsessive behaviors and show strong dysregulation in frontostriatal BDNF function. DAT-KO rats could provide a novel translational model for human diseases involving aberrant DA function and/or mutations affecting DAT or related regulatory mechanisms.SIGNIFICANCE STATEMENT Here, we present a newly developed strain of rats in which the gene encoding the dopamine transporter (DAT) has been disrupted (Dopamine Transporter Knockout rats [DAT-KO rats]). DAT-KO rats display functional hyperdopaminergia accompanied by pronounced spontaneous locomotor hyperactivity. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, and a few other compounds exerting inhibitory action on dopamine-dependent hyperactivity. DAT-KO rats also demonstrate cognitive deficits in working memory and sensorimotor gating tests, less propensity to develop compulsive behaviors, and strong dysregulation in frontostriatal BDNF function. These observations highlight the key role of DAT in the control of brain dopaminergic transmission. DAT-KO rats could provide a novel translational model for human diseases involving aberrant dopamine functions.

Latch and trigger role for R445 in DAT transport explains molecular basis of DTDS.

A recent study reports on five different mutations as sources of dopamine transporter (DAT) deficiency syndrome (DTDS). One of these mutations, R445C, is believed to be located on the intracellular side of DAT distal to the primary (S1) or secondary (S2) sites to which substrate binding is understood to occur. Thus, the molecular mechanism by which the R445C mutation results in DAT transport deficiency has eluded explanation. However, the recently reported X-ray structures of the endogenous amine transporters for dDAT and hSERT revealed the presence of a putative salt bridge between R445 and E428 suggesting a possible mechanism. To evaluate whether the R445C effect is a result of a salt bridge interaction, the mutants R445E, E428R, and the double mutant E428R/R445E were generated. The single mutants R445E and E428R displayed loss of binding and transport properties of the substrate [3H]DA and inhibitor [3H]CFT at the cell surface while the double mutant E428R/R445E, although nonfunctional, restored [3H]DA and [3H]CFT binding affinity to that of WT. Structure based analyses of these results led to a model wherein R445 plays a dual role in normal DAT function. R445 acts as a component of a latch in its formation of a salt bridge with E428 which holds the primary substrate binding site (S1) in place and helps enforce the inward closed protein state. When this salt bridge is broken, R445 acts as a trigger which disrupts a local polar network and leads to the release of the N-terminus from its position inducing the inward closed state to one allowing the inward open state. In this manner, both the loss of binding and transport properties of the R445C variant are explained.

Attentional deficits and altered neuronal activation in medial prefrontal and posterior parietal cortices in mice with reduced dopamine transporter levels.

The executive control function of attention is regulated by the dopaminergic (DA) system. Dopamine transporter (DAT) likely plays a role in controlling the influence of DA on cognitive processes. We examined the effects of DAT depletion on cognitive processes related to attention. Mice with the DAT gene genetically deleted (DAT+/- heterozygotes) were compared to wild type (WT) mice on the Attentional Set-Shifting Task (ASST). Changes in neuronal activity during the ASST were shown with early growth response genes 1 and 2 (egr-1 and egr-2) immunohistochemistry in the medial prefrontal cortex (mPFC) and in the posterior parietal cortex (PPC). Heterozygotes were impaired in tasks that tax reversal learning, attentional-set formation and set-shifting. Densities of egr-2 labeled cells in the mPFC were lower in mutant mice when compared with wild-types in intradimensional shift of attention (IDS), extradimensional shift of attention and extradimensional shift of attention-reversal phases of the ASST task, and in PPC in the IDS phase of the task. The results demonstrate impairments of the areas associated with attentional functions in DAT+/- mice and show that an imbalance of the dopaminergic system has an impact on the complex attention-related executive functions.