Dopamine Transporter Gene Expression Research Articles

Enhanced methamphetamine sensitisation in a rat model of the brain-derived neurotrophic factor Val66Met variant: Sex differences and dopamine receptor gene expression

Brain-derived neurotrophic factor (BDNF) and the Val66Met polymorphism may play a role in the development of psychosis and schizophrenia. The aim of this study was to investigate long-term effects of methamphetamine (Meth) on psychosis-like behaviour and dopamine receptor and dopamine transporter gene expression in a novel rat model of the BDNF Val66Met polymorphism. At the end of a 7-day subchronic Meth treatment, female rats with the Met/Met genotype selectively showed locomotor hyperactivity sensitisation to the acute effect of Meth. Male rats showed tolerance to Meth irrespective of Val66Met genotype. Two weeks later, female Met/Met rats showed increased locomotor activity following both saline treatment or a low dose of Meth, a hyperactivity which was not observed in other genotypes or in males. Baseline PPI did not differ between the groups but the disruption of PPI by acute treatment with apomorphine was absent in Meth-pretreated Met/Met rats. Female Met/Met rats selectively showed down-regulation of dopamine D2 receptor gene expression in striatum. Behavioural effects of MK-801 or its locomotor sensitisation by prior Meth pretreatment were not influenced by genotype. These data suggest a selective vulnerability of female Met/Met rats to short-term and long-term effects of Meth, which could model increased vulnerability to psychosis development associated with the BDNF Val66Met polymorphism.

Interaction of Brain-Derived Neurotrophic Factor with the Effects of Chronic Methamphetamine on Prepulse Inhibition in Mice Is Independent of Dopamine D3 Receptors.

The aim of the present study was to gain a better understanding of the role of brain-derived neurotrophic factor (BDNF) and dopamine D3 receptors in the effects of chronic methamphetamine (METH) on prepulse inhibition (PPI), an endophenotype of psychosis. We compared the effect of a three-week adolescent METH treatment protocol on the regulation of PPI in wildtype mice, BDNF heterozygous mice (HET), D3 receptor knockout mice (D3KO), and double-mutant mice (DM) with both BDNF heterozygosity and D3 receptor knockout. Chronic METH induced disruption of PPI regulation in male mice with BDNF haploinsufficiency (HET and DM), independent of D3 receptor knockout. Specifically, these mice showed reduced baseline PPI, as well as attenuated disruption of PPI induced by acute treatment with the dopamine receptor agonist, apomorphine (APO), or the glutamate NMDA receptor antagonist, MK-801. In contrast, there were no effects of BDNF heterozygosity or D3 knockout on PPI regulation in female mice. Chronic METH pretreatment induced the expected locomotor hyperactivity sensitisation, where female HET and DM mice also showed endogenous sensitisation. Differential sex-specific effects of genotype and METH pretreatment were observed on dopamine receptor and dopamine transporter gene expression in the striatum and frontal cortex. Taken together, these results show a significant involvement of BDNF in the long-term effects of METH on PPI, particularly in male mice, but these effects appear independent of D3 receptors. The role of this receptor in psychosis endophenotypes therefore remains unclear.

Real time monitoring of dopamine release evoked by mitragynine (Kratom): An insight through electrochemical sensor

BackgroundMitragynine, the major indole alkaloid from Mitragyna speciosa has been reported previously to possess abuse liability. However, there are insufficient data suggesting the mechanism through which this pharmacological agent causes addiction. AimsIn this study, we investigated the effects of mitragynine on dopamine (DA) level and dopamine transporter (DAT) expression from the rat’s frontal cortex. MethodsDA level was recorded in the brain samples of animals treated with acute or repeated exposure for 4 consecutive days with either vehicle or mitragynine (1 and 30 mg/kg) using electrochemical sensor. Animals were then decapitated and the brain regions were removed, snap-frozen in liquid nitrogen and immediately stored at −80 °C. DA level was quantified using Enzyme linked immunosorbent assay (ELISA) kits and DAT gene expression was determined using quantitative real time polymerase chain reaction (RT-qPCR). Results/outcomeMitragynine (1 and 30 mg/kg) did not increase DA release following acute treatment, however, after repeated exposure at day 4, mitragynine significantly and dose dependently increased DA release in the frontal cortex. In this study, we also observed a significant increase in DAT mRNA expression at day 4 in group treated with mitragynine (30 mg/kg). Conclusion/interpretationData from this study indicates that mitragynine significantly increased DA release when administered repeatedly, increased in DAT mRNA expression with the highest tested dose (30 mg/kg). Therefore, the rewarding effects observed after mitragynine administration could be due to its ability to increase DA content in certain areas of the brain especially the frontal cortex.

Cannabidiol prevents priming- and stress-induced reinstatement of the conditioned place preference induced by cocaine in mice.

Cocaine dependence is an important problem without any effective pharmacological treatment. Some preclinical studies have suggested that cannabidiol (CBD), a component of the Cannabis sativa plant, could be useful for the treatment of cocaine use disorders. This work aims to evaluate the ability of CBD to reduce priming- and stress-induced reinstatement of the conditioned place preference (CPP) induced by cocaine. Young adult CD-1 male mice were allocated to 10 groups (n = 12/group), conditioned with cocaine (10 mg/kg) and exposed to extinction of CPP (two sessions per week). When extinction was achieved, each group received the corresponding treatment before the reinstatement test. In experiment 1, six groups were used: vehicle+saline (Veh+Sal), 5 mg/kg cocaine alone (Veh+Coc) or with CBD 30 or 60 mg/kg (CBD30+Coc, CBD60+Coc) and CBD alone (CBD30+Sal, CBD60+Sal). In experiment 2, four groups were used: exploration (Veh+Expl), social defeat (Veh+SD) and social defeat with CBD (CBD30+SD and CBD60+SD). Furthermore, the relative gene expression of the dopamine transporter (DAT) in the ventral tegmental area was measured. All mice acquired cocaine CPP and extinguished it after three or four weeks. Only the groups treated with cocaine priming (Veh+Coc) or exposed to social defeat (Veh+SD) showed reinstatement of CPP. Interestingly, CBD itself did not induce reinstatement and blocked the reinstating effects of cocaine priming and social defeat. Furthermore, cocaine priming increased DAT gene expression in the ventral tegmental area and CBD completely reversed this effect. These results suggest that CBD could reduce reinstatement to cocaine seeking after a period of abstinence.

Anti-Inflammatory and Neuromodulatory Effects Induced by Tanacetum parthenium Water Extract: Results from In Silico, In Vitro and Ex Vivo Studies.

Tanacetum parthenium (feverfew) has traditionally been employed as a phytotherapeutic remedy in the treatment of migraine. In this study, a commercial T. parthenium water extract was investigated to explore its anti-inflammatory and neuromodulatory effects. Isolated mouse cortexes were exposed to a K+ 60 mM Krebs-Ringer buffer and treated with T. parthenium water extract. The prostaglandin E2 (PGE2) level, brain-derived neurotrophic factor (BDNF), interleukin-10 (IL-10), and IL-1β gene expression were evaluated in the cortex. The effects on dopamine (DA) release and dopamine transporter (DAT) gene expression were assayed in hypothalamic HypoE22 cells. A bioinformatics analysis was conducted to further investigate the mechanism of action. The extract was effective in reducing cortex PGE2 release and IL-1β gene expression. In the same experimental system, IL-10 and BDNF gene expressions increased, and in HypoE22 cells, the extract decreased the extracellular dopamine level and increased the DAT gene expression due to the direct interaction of parthenolide with the DAT. Overall, the present findings highlight the efficacy of T. parthenium water extract in controlling the inflammatory pathways that occur during cortical-spreading depression. Additionally, the inhibition of the hypothalamic DA release observed in this study further supports the role of dopaminergic pathways as key targets for novel pharmacological approaches in the management of migraine attacks.

Cannabidiol Modulates Behavioural and Gene Expression Alterations Induced by Spontaneous Cocaine Withdrawal.

The aim of this study was to evaluate the effects of cannabidiol (CBD) on the behavioural and gene expression changes in a new animal model of spontaneous cocaine withdrawal. For this purpose, male CD-1 mice were exposed to progressive increasing doses of cocaine for 12days (15 to 60mg/kg/day, i.p.), evaluating spontaneous cocaine withdrawal 6h after the last cocaine administration. The effects of CBD (10, 20, and 40mg/kg, i.p.) were evaluated on cocaine withdrawal-induced alterations in motor activity, somatic signs, and anxiety-like behaviour. Furthermore, gene expression changes in dopamine transporter (DAT) and tyrosine hydroxylase (TH) in the ventral tegmental area, and in cannabinoid receptors 1 (CNR1) and 2 (CNR2) in the nucleus accumbens, were analysed by real-time PCR. The results obtained in the study showed that mice exposed to the spontaneous cocaine withdrawal model presented increased motor activity, somatic withdrawal signs, and high anxiety-like behaviour. Interestingly, the administration of CBD normalized motor and somatic signs disturbances and induced an anxiolytic effect. Moreover, the administration of CBD blocked the increase of DAT and TH gene expression in mice exposed to the cocaine withdrawal, regulated the decrease of CNR1 and induced an additional upregulation of CNR2 gene expression. Thus, this model of spontaneous cocaine withdrawal induces clear behavioural and gene expression changes in mice. Interestingly, CBD alleviates these behavioural and gene expression alterations suggesting its potential for the management of cocaine withdrawal.

Dopamine transporter gene expression within the nucleus accumbens plays important role in the acquisition and reinstatement of ethanol-seeking behavior in mice

Alcoholism and alcohol use disorders are chronically relapsing conditions which is a major problem in treating alcohol addiction. In a previous study we showed that the dopamine transporter (DAT) is implicated in voluntary intake and preference. However, its role in modulating ethanol-associated contextual memory remains largely unknown. In this study we have investigated the role of DAT in ethanol-induced conditioned place preference (EtOH-CPP) acquisition and reinstatement in adult male C57BL/6 mice. For this purpose, we used both loss- and gain-of-function approaches to test the effects of central DAT manipulation on EtOH-CPP. We developed a lentiviral-mediated gene transfer approach to examine whether DAT knockdown (shDAT) or overexpression in the nucleus accumbens (Nacc) is enough to impair EtOH-CPP acquisition and reinstatement. In the first experiment, results showed that DAT knockdown blocked, whereas DAT overexpression, exacerbated the acquisition of EtOH-CPP. In the second experiment and after the EtOH-CPP expression, the mice were subjected to a 14-day extinction trials before drug-induced EtOH-CPP reinstatement was induced by a priming injection of 1 g/kg EtOH. Results indicated that reinstatement of EtOH-CPP was considerably decreased after accumbal shDAT injection. However, DAT overexpression significantly increased EtOH-CPP reinstatement. Finally, and following DAT mRNA quantification using RT-PCR, Pearson’s correlation showed a strong positive relationship between accumbal DAT mRNA and EtOH-CPP acquisition and reinstatement. These results suggest that DAT expression in the Nacc is involved in the acquisition and retrieval of EtOH contextual memory and that blockade of this transporter can decrease the rewarding properties of EtOH.

Reward deficits in an animal model of compulsive eating

BackgroundCompulsive eating, which affects an estimated 15 million adults in the US, can be conceptualized as overeating to relieve a negative emotional state. One hypothesized underlying mechanism is diminished reward. Overeating of palatable diets alter mesolimbic dopamine neurotransmission, likely contributing to the persistence of excessive intake. However, it is not yet known if overeating of palatable food alters reward sensitivity, and whether reward is differentially altered between phases of palatable food access and withdrawal.MethodsIn this study, we investigated sensitivity to d‐Amphetamine, a dopamine releaser, in rats undergoing ad libitum palatable diet cycling, a model of compulsive eating. In this model, animals receive 2 days of access to a highly palatable, chocolate flavored, high‐sucrose diet (‘P Phase’) followed by 5 days of access to a control chow diet (‘C Phase’). In all experiments, we investigated d‐ Amphetamine sensitivity during access to the palatable diet (‘P Phase’) as well as when animals were withdrawn from palatable food (‘C Phase’). To test behavioral and molecular response to d‐ Amphetamine, we used a locomotor response assay, conditioned place preference test, intra‐cranial self‐stimulation paradigm, and in vivo microdialysis. Finally, we tested oral self‐administration of d‐Amphetamine in the home cage. To examine dopaminergic signaling targets, gene expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) were determined using quantitative polymerase chain reaction (qPCR).ResultsDuring the P phase, diet alternated rats showed reduced sensitivity to d‐Amphetamine's stimulating (locomotor assay), rewarding (CPP), and reward‐enhancing effects (ICSS). In diet cycled animals, d‐Amphetamine‐evoked dopamine efflux was blunted. Chow/Palatable rats had higher self‐administration of d‐Amphetamine during the P Phase. Levels of DAT mRNA were increased in the VTA in Chow/Palatable rats during the P Phase.ConclusionsThese results indicate that a history of overconsumption of palatable food causes reward deficits and associated alterations in the mesolimbic pathway that led to increased self‐administration of d‐ Amphetamine.Support or Funding InformationR01‐030425, F31‐DA044664This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Behavioral effects of psychostimulants in mutant mice with cell-type specific deletion of CB2 cannabinoid receptors in dopamine neurons

Activation of the endocannabinoid system modulate dopaminergic pathways that are involved in the effects of psychostimulants including amphetamine, cocaine, nicotine and other drugs of abuse. Genetic deletion or pharmacological activation of CB2 cannabinoid receptor is involved in the modulation of the effects of psychostimulants and their rewarding properties. Here we report on the behavioral effects of psychostimulants in DAT-Cnr2 conditional knockout (cKO) mice with selective deletion of type 2 cannabinoid receptors in dopamine neurons. There was enhanced psychostimulant induced hyperactivity in DAT-Cnr2 cKO mice, but the psychostimulant-induced sensitization was absent in DAT-Cnr2 cKO compared to the WT mice. Intriguingly, lower doses of amphetamine reduced locomotor activity of the DAT-Cnr2 cKO mice. While cocaine, amphetamine and methamphetamine produced robust conditioned place preference (CPP) in both DAT-Cnr2 cKO and WT mice, nicotine at the dose used induced CPP only in the WT but not in the DAT-Cn2 cKO mice. However, pre-treatment with the CB2R selective agonist JWH133, blocked cocaine and nicotine induced CPP in the WT mice. The deletion of CB2Rs in dopamine neurons modified the levels of tyrosine hydroxylase, and reduced the expression of dopamine transporter gene expression in DAT-Cnr2 cKO midbrain region. Taken together, our data suggest that CB2Rs play a role in the modulation of dopamine-related effects of psychostimulants and could be exploited as therapeutic target in psychostimulant addiction and other psychiatric disorders associated with dopamine dysregulation.

Altered Dopamine Synaptic Markers in Postmortem Brain of Obese Subjects.

Dopaminergic signaling in the reward pathway in the brain has been shown to play an important role in food intake and the development of obesity. Obese rats release less dopamine (DA) in the nucleus accumbens (NAc) after food intake, and amphetamine stimulated striatal DA release is reduced in vivo in obese subjects. These studies suggest that DA hypofunction associated with hedonic dysregulation is involved in the pathophysiology of obesity. To identify brain changes in obesity, quantitative measures of DA synaptic markers were compared in postmortem brain tissues of normal weight and obese subjects over a range of increasing body mass indices (BMI). DA transporter (DAT) numbers in the striatum were compared to the relative expression of DAT, tyrosine hydroxylase (TH) and D2 dopamine receptors (DRD2) in midbrain DA neurons. Radioligand binding assays of [3H]WIN35,428 demonstrated that the number of striatal DAT binding sites was inversely correlated with increasing BMI (r = −0.47; p < 0.01). DAT and TH gene expression were significantly decreased in the somatodendritic compartment of obese subjects (p < 0.001), with no significant change in DRD2 compared to normal weight subjects. The reduced density of striatal DAT with corresponding reductions in DAT and TH gene expression in substantia nigra (SN) suggests, that obesity is associated with hypodopaminergic function. A DA reward deficiency syndrome has been suggested to underlie abnormal eating behavior that leads to obesity. Neurobiological changes in presynaptic DA markers demonstrated postmortem in human brain support a link between hedonic DA dysregulation and obesity.

The Abuse Potential of α-Piperidinopropiophenone (PIPP) and α-Piperidinopentiothiophenone (PIVT), Two New Synthetic Cathinones with Piperidine Ring Substituent.

A diversity of synthetic cathinones has flooded the recreational drug marketplace worldwide. This variety is often a response to legal control actions for one specific compound (e.g. methcathinone) which has resulted in the emergence of closely related replacement. Based on recent trends, the nitrogen atom is one of the sites in the cathinone molecule being explored by designer type modifications. In this study, we designed and synthesized two new synthetic cathinones, (1) α-piperidinopropiophenone (PIPP) and (2) α-piperidinopentiothiophenone (PIVT), which have piperidine ring substituent on their nitrogen atom. Thereafter, we evaluated whether these two compounds have an abuse potential through the conditioned place preference (CPP) in mice and self-administration (SA) in rats. We also investigated whether the substances can induce locomotor sensitization in mice following 7 days daily injection and challenge. qRT-PCR analyses were conducted to determine their effects on dopamine-related genes in the striatum. PIPP (10 and 30 mg/kg) induced CPP in mice, but not PIVT. However, both synthetic cathinones were not self-administered by the rats and did not induce locomotor sensitization in mice. qRT-PCR analyses showed that PIPP, but not PIVT, reduced dopamine transporter gene expression in the striatum. These data indicate that PIPP, but not PIVT, has rewarding effects, which may be attributed to its ability to affect dopamine transporter gene expression. Altogether, this study suggests that PIPP may have abuse potential. Careful monitoring of this type of cathinone and related drugs are advocated.

The abuse potential of two novel synthetic cathinones with modification on the alpha-carbon position, 2-cyclohexyl-2-(methylamino)-1-phenylethanone (MACHP) and 2-(methylamino)-1-phenyloctan-1-one (MAOP), and their effects on dopaminergic activity

The recreational use of synthetic cathinones has dramatically increased in recent years, which is partly due to easy accessibility and ability of synthetic cathinones to exert rewarding effects similar to cocaine and methamphetamine. Many synthetic cathinones have already been scheduled in several countries; however, novel and diverse synthetic cathinones are emerging at an unprecedented rate, often outpacing regulatory processes. Recently, designer modifications of the basic cathinone molecule are usually performed on the alpha-carbon position. In this study, we designed and synthesized two novel synthetic cathinones with substituents on alpha-carbon position, [1] 2-cyclohexyl-2-(methylamino)-1-phenylethanone (MACHP), and [2] 2-(methylamino)-1-phenyloctan-1-one (MAOP). Then, we evaluated their rewarding and reinforcing effects through the conditioned place preference (CPP) in mice and self-administration (SA) test in rats. Locomotor activity was also assessed in mice during daily MACHP or MAOP treatment for 7days and drug challenge. qRT-PCR analyses were conducted to determine their effects on dopamine-related genes in the striatum. MACHP and MAOP produced CPP at 10 and 30mg/kg. In the SA test, MACHP (1mg/kg/infusion), but not MAOP, was self-administered. Both MACHP and MAOP induced locomotor sensitization in mice. qRT-PCR analyses showed that MACHP and MAOP reduced dopamine transporter gene expression in the striatum. These data indicate that MACHP and MAOP may have rewarding properties, which might be attributed to their ability to affect the dopaminergic activity. These findings may be useful in predicting the abuse potential and hasten the regulation of future cathinone entities with similar modifications.

Topiramate increases the rewarding properties of cocaine in young-adult mice limiting its clinical usefulness.

Topiramate is an anticonvulsant drug which has been evaluated as a therapeutic option for the treatment of cocaine addiction during the last decade. The purpose of this study was to evaluate the effects of topiramate on the reinforcing actions of cocaine. To this aim, the topiramate-mediated regulation of acquisition and extinction phases of the cocaine conditioned place preference (CPP) was assessed in young-adult mice using three experimental designs. Topiramate (50mg/kg, p.o.) was given as follows: (1) during cocaine (1 and 25mg/kg, i.p.) conditioning sessions (4days) and cocaine (25mg/kg) post-conditioning session; (2) 2weeks before and during cocaine conditioning (25mg/kg); and (3) during extinction of CPP induced by cocaine (25mg/kg). In the first experimental design, changes in tyrosine hydroxylase (TH) and dopamine transporter (DAT) gene expressions were measured in the ventral tegmental area (VTA). Topiramate significantly increased cocaine-induced CPP and delayed or failed to produce extinction after the first cocaine reinstatement extinction in the first and second experiments. Furthermore, treatment with topiramate after place conditioning blocked the extinction of cocaine-induced CPP. TH and DAT gene expression in the VTA was significantly lower both with topiramate alone and in combination with cocaine compared with animals receiving only cocaine. These findings suggest that topiramate increases the rewarding properties of cocaine, at least in part, by regulating dopaminergic signaling in the mesolimbic circuit. Consequently, the results of this study do not support the use of topiramate for the treatment of problems related to cocaine dependence. • Topiramate increases the rewarding properties of cocaine in CPP • Topiramate alters dopaminergic signaling in the mesolimbic circuit • Topiramate delays the extinction of cocaine-induced CPP • TH and DAT gene expression in the VTA decreases with topiramate and/or with cocaine • Results show that it should limit the use of topiramate in cocaine-dependent subjects.

Logopenic syndrome and corticobasal dysfunction in a “benign” type 3 familial cortical myoclonic tremor with epilepsy

Myoclonic epilepsies are frequently related to mitochondrial disorders, lysosomal disorders and proteinopathic neurodegenerative diseases. Familial cortical myoclonic tremor with epilepsy (FCMTE) is a genetic form of myoclonic epilepsy. FCMTE is defined by an autosomal dominant inheritance, adult onset cortical myoclonic tremor and inconstant seizures. Electrophysiologic examinations show cortical hyperexcitability (paroxysmal polyspike and wave activity on EEG with a marked photosensitivity, recording of enlarged cortical components of somatosensory evoked potential, enhanced long-loop reflex (C-reflex), and cortical transients preceding the myoclonic jerks using the jerk-locked back-averaging technique). Type 3 FCMTE (FAME3/FCMTE3) is linked to the locus 5p15.31-p15.1 [1]. No obvious candidate gene (like ion channel subunit gene for example) is localized to this locus. Actually, no mutation has been found and the biological mechanism involved in FCMTE3 remains unclear. Fronto-striatal dysfunction, gait impairment and cognitive disorders appearing after several decades of disease progression were reported in FCMTE3 [2]. The patient was a right-handed 52-year-old man with high level of education. No previous personal medical history or neurodevelopmental disorder was reported. A generalized seizure was reported to have occurred at the age of 25, and with a mild bilateral myoclonus in upper limbs beginning at the age of

Lmx1a regulates dopamine transporter gene expression during ES cell differentiation and mouse embryonic development

Midbrain dopaminergic neurons are implicated in various neurological and psychiatric diseases as well as drug addiction. Thus, the study of their generation and maintenance is pivotal to further our understanding of these disease-underlying mechanisms and development of novel therapeutics. Here, using an embryonic stem cell in vitro differentiation system and mutant dreher mouse, we showed that Lmx1a, an early regulator of midbrain dopamine neural progenitor phenotype specification, is also involved in the regulation of midbrain dopaminergic maturation by regulating gene expression of the dopamine transporter. Forced expression of Lmx1a induced dopamine transporter expression precociously in immature dopaminergic neurons, accompanied by significant increase in specific dopamine uptake. Lmx1a binds to well-conserved sequences in the dopamine transporter promoter region, and this binding sequence directs Lmx1a-dependent activation of reporter gene expression. Furthermore, during mouse embryonic development, dopamine transporter was more severely affected by Lmx1a mutation compared to other dopamine markers such as tyrosine hydroxylase and dopa decarboxylase, again supporting the role of Lmx1a in midbrain dopaminergic maturation in vivo. Thus, this study demonstrates that dopamine transporter is a direct target of Lmx1a and emphasizes a novel role of Lmx1a as one of regulators of mature midbrain dopaminergic neurotransmitter phenotypes.

Decreased Cocaine Motor Sensitization and Self-Administration in Mice Overexpressing Cannabinoid CB2 Receptors

The potential involvement of the cannabinoid CB₂ receptors (CB₂r) in the adaptive responses induced by cocaine was studied in transgenic mice overexpressing the CB₂r (CB₂xP) and in wild-type (WT) littermates. For this purpose, the acute and sensitized locomotor responses to cocaine, conditioned place preference, and cocaine intravenous self-administration were evaluated. In addition, we assessed whether CB₂r were localized in neurons and/or astrocytes, and whether they colocalized with dopamine D1 and D2 receptors (D1Dr and D2Dr). Dopamine (DA) extracellular levels in the nucleus accumbens (NAcc), and gene expression of tyrosine hydroxylase (TH) and DA transporter (DAT) in the ventral tegmental area (VTA), and μ-opioid and cannabinoid CB₁ receptors in the NAcc were also studied in both genotypes. CB₂xP mice showed decreased motor response to acute administration of cocaine (10-20 mg/kg) and cocaine-induced motor sensitization compared with WT mice. CB₂xP mice presented cocaine-induced conditioned place aversion and self-administered less cocaine than WT mice. CB₂r were found in neurons and astrocytes and colocalized with D2Dr in the VTA and NAcc. No significant differences in extracellular DA levels in the NAcc were observed between genotypes after cocaine administration. Under baseline conditions, TH and DAT gene expression was higher and μ-opioid receptor gene expression was lower in CB₂xP than in WT mice. However, both genotypes showed similar changes in TH and μ-opioid receptor gene expression after cocaine challenge independently of the pretreatment received. Importantly, the cocaine challenge decreased DAT gene expression to a lesser extent in cocaine-pretreated CB₂xP than in cocaine-pretreated WT mice. These results revealed that CB₂r are involved in cocaine motor responses and cocaine self-administration, suggesting that this receptor could represent a promising target to develop novel treatments for cocaine addiction.

Increased ethanol intake in prodynorphin knockout mice is associated to changes in opioid receptor function and dopamine transmission

The purpose of this study was to examine the role of the prodynorphin gene in alcohol sensitivity, preference and vulnerability to alcohol consumption. Handling-induced convulsion (HIC) associated to alcohol, alcohol-induced loss of righting reflex (LORR), hypothermic effects in response to acute ethanol challenge, blood ethanol levels (BELs), conditioned place preference, voluntary ethanol consumption and preference, tyrosine hydroxylase (TH), dopamine transporter (DAT) and proenkephalin (PENK) gene expression, and µ-, δ- and κ-opioid agonist-stimulated [S(35) ]- guanosine 5'-triphosphate-binding autoradiography were studied in prodynorphin knockout (PDYN KO) and wild-type (WT) mice. There were no differences in HIC, LORR or the decrease in body temperature in response to acute ethanol challenge between PDYN KO and WT mice. PDYN KO mice presented higher BEL, higher ethanol-conditioned place preference and more ethanol consumption and preference in a two-bottle choice paradigm than WT mice. These findings were associated with lower TH and higher DAT gene expression in the ventral tegmental area and substantia nigra, and with lower PENK gene expression in the caudate-putamen (CPu), accumbens core (AcbC) and accumbens shell (AcbSh) in PDYN KO. The functional activity of the µ-opioid receptor was lower in the CPu, AcbC, AcbSh and cingulate cortex (Cg) of PDYN KO mice. In contrast, δ- and κ-opioid receptor-binding autoradiographies were increased in the CPu and Cg (δ), and in the CPu, AcbC and Cg (κ) of PDYN KO. These results suggest that deletion of the PDYN gene increased vulnerability for ethanol consumption by altering, at least in part, PENK, TH and DAT gene expression, and µ-, δ- and κ-opioid receptor functional activity in brain areas closely related to ethanol reinforcement.

Effect of zinc and paraquat co-exposure on neurodegeneration: Modulation of oxidative stress and expression of metallothioneins, toxicant responsive and transporter genes in rats

Oxidative stress is implicated in Parkinson's disease (PD). Metallothioneins (MT), cytochrome P450 IIE1 (CYP2E1) and glutathione S-transferases alpha4-4 (GSTA4-4) are involved in oxidative stress-mediated damage. Altered dopamine transporter (DAT) and vesicular monoamine transporter-2 (VMAT-2) are also documented in PD. The present study was undertaken to investigate the effect of Zn and PQ co-exposure on neurodegeneration in rats. A significant reduction was observed in spontaneous locomotor activity (SLA), striatal dopamine (DA) levels, tyrosine hydroxylase (TH) immunoreactivity, glutathione reductase (GR) and catalase activity along with increased lipid peroxidation (LPO) and glutathione peroxidase (GPx) activity after Zn and/or PQ exposure. Zn and/or PQ exposure increased gene expression of DAT, CYP2E1, GSTA4-4, MT-I and MT-II, but reduced the expression of VMAT-2. Protein expression analysis of TH, VMAT-2 and DAT showed results similar to those obtained with gene expression study. Zn and PQ co-exposure caused a more pronounced effect than that of individual exposure. The results obtained in this study suggest that, similar to PQ, Zn induced neurodegeneration via alterations in oxidative stress and expression of the above-mentioned genes. However, the effect of Zn+PQ was only slightly higher than that of alone, indicating that probably Zn and PQ follow some different molecular events leading to neurodegeneration.

Maternal deprivation-caused behavioral abnormalities in adult rats relate to a non-methylation-regulated D2 receptor levels in the nucleus accumbens

In this study we investigated the effects of maternal deprivation on adult rats’ spatial learning and memory, exploratory, and limbic activity and their correlations with the gene expression of dopamine transporter (DAT) and dopamine D1, D2, D3 receptors (DRD1, DRD2, DRD3) in the nucleus accumbens (NAc). We further investigated whether DNA methylation is involved in the regulation of DRD2 gene expression. Results from our behavioral tests demonstrated that maternal deprivation significantly decreased the spatial learning and memory ability, exploratory ability, and limbic activity in adult rats, while results from our molecular analysis revealed that the mRNA levels of DAT, DRD1, DRD2, and DRD3 were significantly downregulated in maternally deprived rats in comparison to control animals. Correlation analysis in the maternally deprived rats suggested that downregulated DAT and DRD2 mRNA levels significantly correlated with the amount of time required to find the platform, but only DRD2 significantly correlated with the time spent in target quadrant in the Morris water-maze test; DAT, DRD1 and DRD2 mRNA levels significantly correlated with the number of vertical activity, but only DRD2 mRNA level significantly correlated with total distance in the open field test. Conversely, DRD3 mRNA level did not display any correlation with behavioral changes. The methylated CpG levels in the promoter region of DRD2 gene and the expression of DNA methyltransferases 1 (Dnmt1) and 3α (Dnmt3α) were not significantly changed in maternal deprivation group in comparison to the control group. We concluded that downregulation of DAT, DRD1 and DRD2 gene expression in the NAc is in response to the abnormality in exploratory behaviors; downregulation of DAT and DRD2 expression is in response to the abnormality in spatial learning; downregulation of DRD2 is in response to the abnormality in the spatial memory in maternally deprived rats; whereas DRD3 plays no role in these behaviors. DNA methylation might not be the mechanism to regulate the mRNA level.

Nicotine stimulates transcriptional activity of the human dopamine transporter gene

Nicotine modulates dopaminergic activity in the central nervous system by acting on the reuptake system, including the dopamine transporter (DAT), although precisely remains unclear. Here we investigated the effect of nicotine on the transcriptional regulation of the human DAT (hDAT) gene by conducting luciferase reporter assays. Nicotine enhanced the transcription of hDAT gene constructs in transiently transfected SK-N-SH cells. Hexamethonium, a neuronal (ganglionic) nicotinic acetylcholine receptor antagonist, blocked the action of nicotine. Functional analyses placed the nicotine-responsive region −3.5 to −1.0 kb (from the transcription start site) upstream of the core promotor region. Deletion of intron 1, known as a silencer element of the hDAT gene, abolished nicotine's stimulatory effect. Nicotine failed to stimulate DAT promotor activity in non-neuronal CHO or COS-7 cells or in SK-N-AS cells, another neuronal cell line recently reported as a model for investigating DAT gene expression. These results suggest a nicotinic cholinergic mechanism to be involved in the nicotine-induced up-regulation of DAT gene expression.