The influence of acute dopamine transporter inhibition on manic-, depressive-like phenotypes, and brain oxidative status in adult zebrafish.

Functional changes in dopamine transporter (DAT) are related to various psychiatric conditions, including bipolar disorder (BD) symptoms. In experimental research, the inhibition of DAT induces behavioral alterations that recapitulate symptoms found in BD patients, including mania and depressive mood. Thus, developing novel animal models that mimic BD-related conditions by pharmacologically modulating the dopaminergic signaling is relevant. The zebrafish (Danio rerio) has been considered a suitable vertebrate system for modeling BD-like responses, due to the well-characterized behavioral responses and evolutionarily conservation of the dopaminergic system of this species. Here, we investigate whether GBR 12909, a selective inhibitor of DAT, causes neurobehavioral alterations in zebrafish similar to those observed in BD patients. Behaviors were recorded after a single intraperitoneal (i.p.) administration of GBR 12909 at different doses (3.75, 7.5, 15 and 30 mg/kg). To observe temporal effects on behavior, swim path parameters were measured immediately after the administration period during 30 min. Locomotion, anxiety-like behavior, social preference, aggression, despair-like behavior, and oxidative stress-related biomarkers in the brain were measured 30 min post administration. GBR 12909 induced prominent effects on locomotor activity and vertical exploration during the 30-min period. Hyperactivity was observed in GBR 30 group after 25 min, while all doses markedly reduced vertical drifts. GBR 12909 elicited hyperlocomotion, anxiety-like behavior, decreased social preference, aggression, and induced depressive-like behavior in a behavioral despair task. Depending on the dose, GBR 12909 also decreased SOD activity and TBARS levels, as well as increased GR activity and NPSH content. Collectively, our novel findings show that a single GBR 12909 administration evokes neurobehavioral changes that recapitulate manic- and depressive-like states observed in rodents, fostering the use of zebrafish models to explore BD-like responses in translational neuroscience research.

Exploring transgenerational inheritance in epigenotypes of DAT heterozygous rats: Circadian anomalies and attentional vulnerability.

Dopamine (DA) is mainly involved in locomotor activity, reward processes and maternal behaviors. Rats with KO gene for dopamine transporter (DAT), coding for a truncated DAT protein, are in hyperdopaminergic conditions and thus develop stereotyped behaviors and hyperactivity. Our aim was to test the prior transgenerational modulation of wild and truncated alleles as expressed in heterozygous DAT rats: specifically, we addressed the possible sequelae due to genotype and gender of the ancestors, with regard to behavioral differences in F1, F2, F3 rats. We studied non-classical DAT heterozygotes (HETs) based on two specular lines, with putative grand-maternal vs. grand-paternal imprinting. MAT females (F1; offspring of KO male and WT female) mated with a KO male to generate MIX offspring (F2). Specularly, PAT females (F1; offspring of KO female and WT male) mated with a KO male to generate PIX offspring (F2). Similarly to PAT, we obtained MUX (F2; HET offspring of MAT sire and KO dam); we also observed the F3 (MYX: HET offspring of KO male and MUX female, thus with DAT-KO maternal grandmother like also for PIX). We studied their circadian cycle of locomotor activity and their behavior in the elevated-plus-maze (EPM). Locomotor hyper-activity occurs in F1, the opposite occurs in F2, with MYX rats appearing undistinguishable from WT ones. Open-arm preference emerged in PIX and MIX rats. Only MAT and MYX rats showed a significant vulnerability for ADHD-like inattentive symptoms (duration of rearing in the EPM; Viggiano et al., 2002). A risk-taking profile is evident in the F2 phenotype, while inattentiveness from F1 progeny tends to be transferred to F3. We hypothesize that DAT-related phenotypes result from effective inheritance through pedigree of imprints that are dependent on grandparents, suggesting a protective role for gestation within a hyperdopaminergic uterus. For major features, similar odd (F1, F3) generations appear opposed to even (F2) ones; for minor specific features, the phenotype transfer may affect the progenies with a male but not a female DAT-KO ancestor.

Generation of Dopamine Transporter (DAT)-mCherry Knock-in Rats by CRISPR-Cas9 Genome Editing.

Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.

Amphetamine Induces Sex-Dependent Loss of the Striatal Dopamine Transporter in Sensitized Mice.

Dopamine transporter (DAT) controls dopamine signaling in the brain through the reuptake of synaptically released dopamine. DAT is a target of abused psychostimulants such as amphetamine (Amph). Acute Amph administration induces transient DAT endocytosis, which, among other Amph effects on dopaminergic neurons, elevates extracellular dopamine. However, the effects of repeated Amph abuse, leading to behavioral sensitization and drug addiction, on DAT are unknown. Hence, we developed a 14 d Amph-sensitization protocol in knock-in mice expressing HA-epitope-tagged DAT (HA-DAT) and investigated the effects of Amph challenge on sensitized HA-DAT animals. The Amph challenge resulted in the highest locomotor activity on Day 14 in both sexes, which was sustained for 1 h in male but not female mice. Strikingly, significant (by 30-60%) loss of the HA-DAT protein in the striatum was caused by the Amph challenge of sensitized males but not females. Amph also reduced V max of dopamine transport in the striatal synaptosomes of males without changing K m values. Consistently, immunofluorescence microscopy revealed a significant increase of HA-DAT colocalization with the endosomal protein VPS35 only in Amph-challenged males. Amph-induced loss of striatal HA-DAT in sensitized mice was blocked by chloroquine, vacuolin-1, and inhibitor of Rho-associated kinases ROCK1/2, indicative of the involvement of endocytic trafficking in the DAT protein loss. Interestingly, an apparent degradation of HA-DAT protein was observed in the nucleus accumbens and not in the dorsal striatum. We propose that Amph challenge in sensitized mice triggers Rho-mediated endocytosis and post-endocytic trafficking of DAT in a brain-region-specific and sex-dependent manner.

Early-life perfluorooctanoic acid exposure disrupts the function of dopamine transporter protein with glycosylation changes implicating the links between decreased dopamine levels and disruptive behaviors in larval zebrafish.

Exposure to perfluorooctanoic acid (PFOA) during early embryonic development is associated with the increased risk of developmental neurotoxicity and neurobehavioral disorders in children. In our previous study, we demonstrated that exposure to PFOA affected locomotor activity and disrupted dopamine-related gene expression in zebrafish larvae. Consequently, we continue to study the dopaminergic system with a focus on dopamine levels and dopamine's effect on behaviors in relation to PFOA exposure. In the present study, we found a decrease in dopamine levels in larval zebrafish. We studied the dopamine transporter (DAT) protein, which is responsible for regulating dopamine levels through the reuptake of dopamine in neuronal cells. We demonstrated that exposure to PFOA disrupted the glycosylation process of DAT, inhibited its uptake function, and induced endoplasmic reticulum (ER) stress in dopaminergic cells. Besides, we conducted a light-dark preference test on larval zebrafish and observed anxiety/depressive-like behavioral changes following exposure to PFOA. Dopamine is one of the most prominent neurotransmitters that significantly influences human behavior, with low dopamine levels being associated with impairments such as anxiety and depression. The anxiety-like response in zebrafish larvae exposure to PFOA implies the link with the reduced dopamine levels. Taken together, we can deduce that glycosylation changes in DAT lead to dysfunction of DAT to regulate dopamine levels, which in turn alters behavior in larval zebrafish. Therefore, alternation in dopamine levels may play a pivotal role in the development of anxiety/depressive-like behavioral changes induced by PFOA.

Leaky lessons learned: Efflux prone dopamine transporter variant reveals sex and circuit specific contributions of D2 receptor signalling to neuropsychiatric disease.

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.

Gene Interaction of Dopaminergic Synaptic Pathway Genes in Attention-Deficit Hyperactivity Disorder: a Case-Control Study in Chinese Children.

Attention-deficit hyperactivity disorder is a highly inherited neurodevelopmental disorder. Previous genetic research has linked ADHD to certain genes in the dopaminergic synaptic pathway. Nonetheless, research on this relationship has produced varying results across various populations. China is a multi-ethnic country with its own unique genetic characteristics. Therefore, such a population can provide useful information about the relationship between gene polymorphisms in dopaminergic synaptic pathways and ADHD. This study looked at the genetic profiles of 284 children in China's Xinjiang. In total, 142 ADHD children and 142 control subjects were enrolled. Following the extraction of DNA from oral mucosal cells, 13 SNPs for three candidate genes (SLC6A3, DRD2, and GRIN2B) in the dopaminergic synaptic pathway of ADHD were screened. Based on the results of single nucleotide polymorphism (SNP) analyses, we found that the DRD2 gene variants rs6277 and rs6275, and the SLC6A3 gene variant rs2652511, were significantly associated with ADHD in boys and girls, respectively, after adjusting for false discovery rate (FDR) in terms of allele frequencies. Furthermore, our generalized multifactorial downscaling approach identified a significant association between rs6275 and rs1012586. These findings suggest that DRD2 and SLC6A3 genes have a crucial role in ADHD susceptibility. Additionally, we observed that the interaction between GRIN2B and DRD2 genes may contribute to the susceptibility of Chinese children with ADHD.

Hyperdopaminergia in rats is associated with reverse effort-cost dependent performance.

BACKGROUND: Dopamine is implicated in the effort-based control of motivational processes; however, whether tonic dopamine regulates the effort-cost impact on motivation, is still debated. AIMS: The rats lacking the dopamine transporter (DAT), which have dramatically increased levels of the synaptic dopamine, were used in the present study to elucidate the role of the synaptic dopamine in motivational processes. METHODS: To study the reward-related processes, the progressive ratio 3 (PR3) operant schedule of food reinforcement (the ratio increases by 3 after each earned reinforcer) was performed in adult male rats (DAT knockouts (DAT-KO), heterozygotes (DAT-HT) and wild-types (DAT-WT)). RESULTS: During the PR3 session, the response rate of DAT-KO rats was gradually increased following the augmented required number of responses. In contrast, the local response rate of DAT-WT and DAT-HT decreased. d-Amphetamine sulfate salt (3 mg/kg, i.p.) altered the local response rate dynamics in DAT-WT, which became similar to that of DAT-KO. Interestingly, the reduction in response rate at low effort demands was associated with decreased rate of entries into the magazine tray in DAT-WT rats treated with amphetamine (3 mg/kg) but not in DAT-KO rats. CONCLUSIONS: Our results suggest that the elevated tonic synaptic dopamine can strongly affect motivation/effort-cost relation in rodents.

Dopamine-Depleted Dopamine Transporter Knockout (DDD) Mice: Dyskinesia with L-DOPA and Dopamine D1 Agonists.

L-DOPA is the mainstay of treatment for Parkinson's disease (PD). However, over time this drug can produce dyskinesia. A useful acute PD model for screening novel compounds for anti-parkinsonian and L-DOPA-induced dyskinesia (LID) are dopamine-depleted dopamine-transporter KO (DDD) mice. Treatment with α-methyl-para-tyrosine rapidly depletes their brain stores of DA and renders them akinetic. During sensitization in the open field (OF), their locomotion declines as vertical activities increase and upon encountering a wall they stand on one leg or tail and engage in climbing behavior termed "three-paw dyskinesia". We have hypothesized that L-DOPA induces a stereotypic activation of locomotion in DDD mice, where they are unable to alter the course of their locomotion, and upon encountering walls engage in "three-paw dyskinesia" as reflected in vertical counts or beam-breaks. The purpose of our studies was to identify a valid index of LID in DDD mice that met three criteria: (a) sensitization with repeated L-DOPA administration, (b) insensitivity to a change in the test context, and (c) stimulatory or inhibitory responses to dopamine D1 receptor agonists (5 mg/kg SKF81297; 5 and 10 mg/kg MLM55-38, a novel compound) and amantadine (45 mg/kg), respectively. Responses were compared between the OF and a circular maze (CM) that did not hinder locomotion. We found vertical counts and climbing were specific for testing in the OF, while oral stereotypies were sensitized to L-DOPA in both the OF and CM and responded to D1R agonists and amantadine. Hence, in DDD mice oral stereotypies should be used as an index of LID in screening compounds for PD.

Long-Lasting Epigenetic Changes in the Dopamine Transporter in Adult Animals Exposed to Amphetamine during Embryogenesis: Investigating Behavioral Effects.

The dopamine transporter (DAT) is an integral member of the dopaminergic system and is responsible for the release and reuptake of dopamine from the synaptic space into the dopaminergic neurons. DAT is also the major target of amphetamine (Amph). The effects of Amph on DAT have been intensively studied; however, the mechanisms underlying the long-term effects caused by embryonal exposure to addictive doses of Amph remain largely unexplored. As in mammals, in the nematode C. elegans Amph causes changes in locomotion which are largely mediated by the C. elegans DAT homologue, DAT-1. Here, we show that chronic embryonic exposures to Amph alter the expression of DAT-1 in adult C. elegans via long-lasting epigenetic modifications. These changes are correlated with an enhanced behavioral response to Amph in adult animals. Importantly, pharmacological and genetic intervention directed at preventing the Amph-induced epigenetic modifications occurring during embryogenesis inhibited the long-lasting behavioral effects observed in adult animals. Because many components of the dopaminergic system, as well as epigenetic mechanisms, are highly conserved between C. elegans and mammals, these results could be critical for our understanding of how drugs of abuse initiate predisposition to addiction.

Association of protein distribution and gene expression revealed by positron emission tomography and postmortem gene expression in the dopaminergic system of the human brain.

PURPOSE: The topological distribution of dopamine-related proteins is determined by gene transcription and subsequent regulations. Recent research strategies integrating positron emission tomography with a transcriptome atlas have opened new opportunities to understand the influence of regulation after transcription on protein distribution. Previous studies have reported that messenger (m)-RNA expression levels spatially correlate with the density maps of serotonin receptors but not with those of transporters. This discrepancy may be due to differences in regulation after transcription between presynaptic and postsynaptic proteins, which have not been studied in the dopaminergic system. Here, we focused on dopamine D1 and D2/D3 receptors and dopamine transporters and investigated their region-wise relationship between mRNA expression and protein distribution. METHODS: We examined the region-wise correlation between regional binding potentials of the target region relative to that of non-displaceable tissue (BPND) values of 11C-SCH-23390 and mRNA expression levels of dopamine D1 receptors (D1R); regional BPND values of 11C-FLB-457 and mRNA expression levels of dopamine D2/D3 receptors (D2/D3R); and regional total distribution volume (VT) values of 18F-FE-PE2I and mRNA expression levels of dopamine transporters (DAT) using Spearman's rank correlation. RESULTS: We found significant positive correlations between regional BPND values of 11C-SCH-23390 and the mRNA expression levels of D1R (r = 0.769, p = 0.0021). Similar to D1R, regional BPND values of 11C-FLB-457 positively correlated with the mRNA expression levels of D2R (r = 0.809, p = 0.0151) but not with those of D3R (r = 0.413, p = 0.3095). In contrast to D1R and D2R, no significant correlation between VT values of 18F-FE-PE2I and mRNA expression levels of DAT was observed (r = -0.5934, p = 0.140). CONCLUSION: We found a region-wise correlation between the mRNA expression levels of dopamine D1 and D2 receptors and their respective protein distributions. However, we found no region-wise correlation between the mRNA expression levels of dopamine transporters and their protein distributions, indicating different regulatory mechanisms for the localization of pre- and postsynaptic proteins. These results provide a broader understanding of the application of the transcriptome atlas to neuroimaging studies of the dopaminergic nervous system.

Antioxidant system alterations and physiological characteristics of neonatal and juvenile DAT-KO rats.

Dopamine transporter knockout (DAT-KO) rats represent a valuable rodent model for studying the molecular and phenotypical outcomes of the effects of excessive dopamine accumulation in the synaptic cleft and the prolonged action of dopamine on neurons. Animals with DAT deficiency are characterized by hyperactivity, stereotypy, cognitive deficits, and impairments in behavioral and biochemical indicators. Several key pathophysiological mechanisms are known to be common to psychiatric, neurodegenerative, metabolic, and other diseases. Among these mechanisms, oxidative stress systems play a particularly important role. One of the main antioxidant systems in the brain is glutathione: specifically, glutathione S-transferase, glutathione reductase, and catalase play a significant role in the regulation of vital oxidative processes, and their dysfunction has been shown in Parkinson's disease, Alzheimer's disease, and other neurodegenerative diseases. The current study aimed to analyze the dynamics of the activity levels of glutathione reductase and glutathione S-transferase in erythrocytes, as well as catalase in the blood plasma, of DAT-deficient, homo- and heterozygous, neonatal and juvenile rats (both male and female). Their behavioral and physiological parameters were evaluated at the age of 1.5 months. For the first time, changes in physiological and biochemical parameters were shown in DAT-KO rats at 1.5 months of postnatal life. The key role of glutathione S-transferase, glutathione reductase, and catalase in the regulation of oxidative stress in DAT-KO rats at the 5th week of life was demonstrated. A positive effect of a slightly increased dopamine level on memory function was shown in DAT-heterozygous animals.

Dopamine Transporter Knockout Rats Display Epigenetic Alterations in Response to Cocaine Exposure.

(1) Background: There is an urgent need for effective treatments for cocaine use disorder (CUD), and new pharmacological approaches targeting epigenetic mechanisms appear to be promising options for the treatment of this disease. Dopamine Transporter (DAT) transgenic rats recently have been proposed as a new animal model for studying susceptibility to CUD. (2) Methods: DAT transgenic rats were treated chronically with cocaine (10 mg/kg) for 8 days, and the expression of epigenetic modulators, Lysine Demethylase 6B (KDM6B) and Bromodomain-containing protein 4 (BRD4), was examined in the prefrontal cortex (PFC). (3) Results: We show that only full knockout (KO) of DAT impacts basal levels of KDM6B in females. Additionally, cocaine altered the expression of both epigenetic markers in a sex- and genotype-dependent manner. In response to chronic cocaine, KDM6B expression was decreased in male rats with partial DAT mutation (HET), while no changes were observed in wild-type (WT) or KO rats. Indeed, while HET male rats have reduced KDM6B and BRD4 expression, HET female rats showed increased KDM6B and BRD4 expression levels, highlighting the impact of sex on epigenetic mechanisms in response to cocaine. Finally, both male and female KO rats showed increased expression of BRD4, but only KO females exhibited significantly increased KDM6B expression in response to cocaine. Additionally, the magnitude of these effects was bigger in females when compared to males for both epigenetic enzymes. (4) Conclusions: This preliminary study provides additional support that targeting KDM6B and/or BRD4 may potentially be therapeutic in treating addiction-related behaviors in a sex-dependent manner.

Dopamine Transporter Deficiency Syndrome (DTDS): Expanding the Clinical Phenotype and Precision Medicine Approaches.

Infantile parkinsonism-dystonia due to dopamine transporter deficiency syndrome (DTDS) is an ultrarare childhood movement disorder caused by biallelic loss-of-function mutations in the SLC6A3 gene. Advances in genomic analysis have revealed an evolving spectrum of SLC6A3-related neurological and neuropsychiatric disorders. Since the initial clinical and genetic characterisation of DTDS in 2009, there have been thirty-one published cases with a variety of protein-truncating variants (nonsense variants, splice-site changes, and deletions) and missense changes. Amino acid substitutions result in mutant proteins with impaired dopamine transporter function due to reduced transporter activity, impaired dopamine binding, reduced cell-surface expression, and aberrant posttranslational protein modification with impaired glycosylation. In this review, we provide an overview of the expanding clinical phenotype of DTDS and the precision therapies in development, including pharmacochaperones and gene therapy.

Patterns of ethanol intake in male rats with partial dopamine transporter deficiency.

Mesolimbic dopamine signaling plays a major role in alcohol and substance use disorders as well as comorbidities such as anxiety and depression. Growing evidence suggests that alcohol drinking is modulated by the function of the dopamine transporter (DAT), which tightly regulates extracellular dopamine concentrations. Adult male rats on a Wistar Han background (DAT+/+) and rats with a partial DAT deletion (DAT+/-) were used in this study. First, using fast-scan cyclic voltammetry in brain slices containing the nucleus accumbens core from ethanol-naïve subjects, we measured greater evoked dopamine concentrations and slower dopamine reuptake in DAT+/- rats, consistent with increased dopamine signaling. Next, we measured ethanol drinking using the intermittent access two-bottle choice paradigm (20% v/v ethanol vs. water) across 5 weeks. DAT+/- rats voluntarily consumed less ethanol during its initial availability (the first 30 min), especially after longer periods of deprivation. In addition, DAT+/- males consumed less ethanol that was adulterated with the bitter tastant quinine. These findings suggest that partial DAT blockade and concomitant increase in brain dopamine levels has potential to reduce drinking and ameliorate alcohol use disorder (AUD).

Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca2.

Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on activation of Ca2+-dependent pathways, but the mechanism underlying reverse transport via endogenously expressed DAT is still unclear. Here, to enable concurrent visualization by live imaging of extracellular DA dynamics and cytosolic Ca2+ levels, we employ the fluorescent Ca2+ sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRABDA1H expressed in cocultured "sniffer" cells. In the presence of the Na+-channel blocker tetrodotoxin to prevent exocytotic DA release, AMPH induced in the cultured neurons a profound dose-dependent efflux of DA that was blocked both by inhibition of DAT with cocaine and by inhibition of the vesicular monoamine transporter-2 with Ro-4-1284 or reserpine. However, the AMPH-induced DA efflux was not accompanied by an increase in cytosolic Ca2+ and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca2+. The independence of cytosolic Ca2+ was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca2+ without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca2+ spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca2+ in an autoreceptor-dependent manner regardless of the apparent independence of Ca2+ for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca2+ but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT.

Reduced Striatal Dopamine Transporter Availability and Heightened Response to Natural and Pharmacological Stimulation in CCK-1R-Deficient Obese Rats.

Alterations in dopamine neurotransmission are associated with obesity and food preferences. Otsuka Long-Evans Tokushima Fatty (OLETF) rats that lack functional cholecystokinin receptor type-1 (CCK-1R), due to a natural mutation, exhibit impaired satiation, are hyperphagic, and become obese. In addition, compared to lean control Long-Evans Tokushima (LETO) rats, OLETF rats have pronounced avidity for over-consuming palatable sweet solutions, have greater dopamine release to psychostimulants, reduced dopamine 2 receptor (D2R) binding, and exhibit increased sensitivity to sucrose reward. This supports altered dopamine function in this strain and its general preference for palatable solutions such as sucrose. In this study, we examined the relationship between OLETF's hyperphagic behavior and striatal dopamine signaling by investigating basal and amphetamine stimulated motor activity in prediabetic OLETF rats before and after access to sucrose solution (0.3 M) compared to non-mutant control LETO rats, as well as availability of dopamine transporter (DAT) using autoradiography. In the sucrose tests, one group of OLETF rats received ad libitum access to sucrose while the other group received an amount of sucrose equal to that consumed by the LETO. OLETFs with ad libitum access consumed significantly more sucrose than LETOs. Sucrose exerted a biphasic effect on basal activity in both strains, i.e., reduced activity for 1 week followed by increased activity in weeks 2 and 3. Basal locomotor activity was reduced (-17%) in OLETFs prior to sucrose, compared to LETOs. Withdrawal of sucrose resulted in increased locomotor activity in both strains. The magnitude of this effect was greater in OLETFs and the activity was increased in restricted compared to ad-libitum-access OLETFs. Sucrose access augmented AMPH-responses in both strains with a greater sensitization to AMPH during week 1, an effect that was a function of the amount of sucrose consumed. One week of sucrose withdrawal sensitized AMPH-induced ambulatory activity in both strains. In OLETF with restricted access to sucrose, withdrawal resulted in no further sensitization to AMPH. DAT availability in the nucleus accumbens shell was significantly reduced in OLETF compared with aged-matched LETO. Together, these findings show that OLETF rats have reduced basal DA transmission and a heightened response to natural and pharmacological stimulation.

Hypomethylation of the dopamine transporter (DAT) gene promoter is associated with hyperphagia-related behavior in Prader-Willi syndrome: A case-control study.

Prader-Willi syndrome (PWS), a neurodevelopmental disorder based on the loss of paternally derived but maternally imprinted genes on chromosome 15q11-13, is typically associated with hyperphagia-related behavior leading to massive obesity. Recently, there has been increasing evidence for dysregulated expression patterns of genes outside the PWS locus that influence the behavioral phenotype and for alterations in the dopaminergic system associated with weight regulation in PWS. In this study, we investigated the epigenetic regulation of the promoter regions of the dopamine transporter (DAT) and dopamine receptor D2 (DRD2) genes and their association with hyperphagia-related behavior in PWS. Methylation of the DAT and DRD2 promoter regions was examined by DNA bisulfite sequencing in 32 individuals with PWS and compared with a control group matched for sex, age, and body mass index (BMI). Hyperphagia-related behavior was assessed using the Hyperphagia Questionnaire for Clinical Trials (HQ-CT). Analysis by linear mixed models revealed a significant effect of factor group on mean DAT promoter methylation rate with decreased mean methylation in PWS (7.3 ± 0.4%) compared to controls (18.8 ± 0.6%), p < 0.001. In the PWS group, we further identified effects of HQ-CT score and BMI on DAT promoter methylation. Although also statistically significantly different (8.4 ± 0.2 in PWS, 10.5 ± 0.3 in controls, p < 0.001), DRD2 promoter methylation visually appeared to be evenly distributed between groups, raising concerns regarding a biological effect. Here, we provide evidence for altered epigenetic regulation of the DAT gene in PWS, which is associated with PWS-typical hyperphagia-related behaviors.

Epigenetic Analysis of the Dopamine Transporter Gene DAT1 with a Focus on Personality Traits in Athletes.

Human phenotypes (traits) are determined by the selective use of a person's unique genotype (DNA sequence), following exposure to environmental stimuli, such as exercise. Inducing profound changes in epigenetics may be an underlying factor of the beneficial effects of exercise. This study aimed to investigate the association between methylation in the promoter region of the DAT1 gene and personality traits measured by the NEO-FFI questionnaire in a group of athletes. The study group included 163 athletes, and the control group consisted of 232 non-athletes. The obtained results show several significant differences between the studied groups of subjects. The Extraversion scale and the Conscientiousness scale results of the NEO-FFI are significantly higher in the group of athletes compared to controls. The total methylation and the number of methylated islands in the promoter region of the DAT1 gene are higher in the study group. Pearson's linear correlation between the total methylation, the number of methylated islands and the NEO-FFI shows significant results for the Extraversion and Agreeability scales. The total methylation and the number of methylated islands in the promoter region of the DAT1 gene are higher in the study group. Pearson's linear correlation between the total methylation, the number of methylated islands and the NEO-FFI shows significant results for the Extraversion and Agreeability scales. Our analysis of the methylation status of individual CpG sites revealed a new direction of research into the biological aspects of regulating dopamine release and personality traits in people practicing sports.

Altered responsiveness to glutamatergic modulation by MK-801 and to repeated stress of immune challenge in female dopamine transporter knockout rats.

Chronic stress is a key factor in psychiatric and neurological disorders often worsening disease symptoms. In this study, a unique animal model, the dopamine transporter knockout (DAT-KO) rat exhibiting behavioral signs resembling those occurring in mania, schizophrenia, attention deficit hyperactivity disorder, and obsessive-compulsive disorder was used. We have tested the hypothesis that the hyperdopaminergic state in DAT-KO rats (i) modulates behavioral response to the NMDA antagonist MK-801 (dizocilpine) and (ii) leads to abnormal endocrine and immune activation under subchronic stress induced by an immune challenge. Glutamatergic modulation with MK-801 induced a different behavioral pattern. While the WT rats responded to MK-801 injection with a robust rise in their locomotor activity, the hyperactive DAT-KO rats exhibited reduced locomotion. Signs of chronic stress including increased basal corticosterone and aldosterone but blunted anxiety were demonstrated in rats lacking the DAT. Repeated injections of increasing doses of lipopolysaccharide (LPS, 5 days) did not modify plasma prolactin concentrations which were however significantly lower in DAT-KO than in WT rats. Concentrations of plasma high mobility group box 1 (HMGB1) protein were significantly higher in LPS-treated DAT-KO than in WT rats. The gene expression of interleukin-6 in the anterior pituitary increased under the stress induced by the immune challenge in the WT but not the DAT-KO rats. The most evident differences between the genotypes were revealed in the spleen. The splenic gene expression of interleukin-1ß, interleukin-6, and HMGB1 was lower and that of ferritin was higher in DAT-KO compared to WT rats. Obtained results emphasize the functional interaction of the endocrine and immune systems with monoamine and glutamatergic neurotransmission in the mechanisms leading to behavioral alterations and psychiatric disorders associated with dopamine dysfunction.