Dopamine Transporter mRNA Research Articles

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.

Proliferation and committed differentiation into dopamine neurons of neural stem cells induced by the active ingredients of radix astragali.

Neural stem cells (NSCs) are important cellular sources of transplantation therapies for Parkinson’s disease. This study aimed to determine the effects of extracts of radix astragali on the proliferation and differentiation into dopamine (DA) neurons in NSCs. NSCs were dealt with astragaloside IV (ASI), astragalus polysaccharide (APS), and astraisoflavan (ASF), the main active ingredients of radix astragali. First, the results from cell-count kit-8 (CCK-8) assay showed that ASI, ASF, and APS had positive effects on the proliferation of NSCs. Next, we also confirmed the effects of ASI, APS, and ASF on BrdU and nestin by immunocytochemistry. Moreover, results from quantitative RT-PCR showed ASI, APS, and ASF could promote the expressions of tyrosine hydroxylase and dopamine transporter mRNA, which are specifically expressed in DA neurons. Simultaneously, sonic hedgehog (Shh), orphan nuclear hormone 1 (Nurr1), and pituitary homeobox 3 (Ptx3) are considered to motivate the formation of DA neurons. Our result showed ASI, APS, and ASF can also promote the expressions of Shh, Nurr1, and Ptx3 mRNAs. In conclusion, our study verifies that the active ingredients of radix astragali can promote the proliferation of NSCs and induce NSC differentiation toward DA neurons in vitro. These phenomena may occur through upregulation of Shh, Nurr1, and Ptx3 in the process of drug treatment.

Tyrosine Hydroxylase, Vesicular Monoamine Transporter and Dopamine Transporter mRNA Expression in Nigrostriatal Tissue of Rats with Pedunculopontine Neurotoxic Lesion.

Background: The degeneration of the pedunculopontine nucleus (PPN) precedes the degeneration of the nigral cells in the pre-symptomatic stages of Parkinson’s disease (PD). Although the literature recognizes that a lesion of the PPN increases the vulnerability of dopaminergic cells, it is unknown if this risk is associated with the loss of capability of handling the dopaminergic function. Methods: In this paper, the effects of a unilateral neurotoxic lesion of the PPN in tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) mRNA expression in nigrostriatal tissue were evaluated. Three experimental groups were organized: non-treated rats, NMDA-lesioned rats and Sham-operated rats. Results: Seven days after the PPN lesion, in nigral tissue, TH mRNA expression was higher in comparison with control groups (p < 0.05); in contrast, VMAT2 mRNA expression showed a significant decrease (p < 0.01). DAT mRNA expression showed a significant decrease (p < 0.001) in the striatal tissue. Comparing nigral neuronal density of injured and control rats revealed no significant difference seven days post-PPN injury. Conclusions: Findings suggest that the PPN lesion modifies the mRNA expression of the proteins associated with dopaminergic homeostasis at nigrostriatal level. It could represent vulnerability signals for nigral dopaminergic cells and further increase the risk of degeneration of these cells.

Safranal-promoted differentiation and survival of dopaminergic neurons in an animal model of Parkinson’s disease

Context: Safranal (SAF) is verified to have potential effects in promoting nerve growth.Objectives: This study verifies the role of SAF in promoting dopaminergic neurons growth in vitro and in vivo.Material and methods: Rat neural stem cells (NSC) were treated with 1, 20, or 100 ng/mL of SAF, and the expression levels of tyrosine hydroxylase (TH) and dopamine transporter (DAT) were assayed by flow cytometry and real-time PCR and the secretion of dopamine (DA) was assayed by ELISA. Then, 2 × 106 cells of SAF-treated NSC was administrated into PD rat models induced by 6-OHDA. The differentiation and survival of dopaminergic neurons was identified by fluorescence microscope and TH+ cells by immunostaining and DA secretion by ELISA at week 2 and week 4, respectively.Results: After being treated with SAF at 20 and 100 ng/mL for 1 week, TH and DAT positive rates increased 1.4- and 1.7-fold (p < 0.01, respectively). TH and DAT mRNA also increased 8.05- and 4.41-fold, respectively. And the release of DA statistically increased 1.5-fold (p < 0.01). In vivo, the number of rotations decreased to 4.33 ± 0.97 rpm (p < 0.01) and the survival rates increased to 77.66 ± 7.87% (p < 0.05) at week 4 after transplantation of SAF-treated NSC. Moreover, the transplanted cells increased three-fold, TH fluorescence density increased four-fold and DA releases increased 1.4-fold (p < 0.01) at week 4 after transplantation.Conclusions: SAF promoted the production of functional DA cells and alleviated PD, which may contribute to a new therapy for PD patients.

A new synthetic drug 5-(2-aminopropyl)indole (5-IT) induces rewarding effects and increases dopamine D1 receptor and dopamine transporter mRNA levels

In recent years, there has been a marked increase in the use of recreational synthetic psychoactive substances, which is a cause of concern among healthcare providers and legal authorities. In particular, there have been reports on the misuse of 5-(2-aminopropyl)indole (5-API; 5-IT), a new synthetic drug, and of fatal and non-fatal intoxication. Despite these reports, little is known about its psychopharmacological effects and abuse potential. Here, we investigated the abuse potential of 5-IT by evaluating its rewarding and reinforcing effects through conditioned place preference (CPP) (1, 10, and 30 mg/kg, i.p.) in mice and self-administration test (0.1, 0.3, 1, and 3 mg/kg/inf., i.v.) in rats. We also examined whether 5-IT (1, 3, and 10 mg/kg, i.p.) induces locomotor sensitization in mice following a 7-day treatment and drug challenge. Then, we explored the effects of 5-IT (10 mg/kg, i.p.) on dopamine-related genes in the striatum, prefrontal cortex (PFC), and substantia nigra pars compacta (SNc)/ventral tegmental (VTA) of mice by quantitative real-time polymerase chain reaction. 5-IT produced CPP in mice but was not reliably self-administered by rats. 5-IT also induced locomotor sensitization following repeated administration and drug challenge. Moreover, 5-IT increased mRNA levels of dopamine D1 receptor in the striatum and PFC and dopamine transporter in the SNc/VTA of mice. These results indicate that 5-IT has psychostimulant and rewarding properties, which may be attributed to its ability to affect the dopaminergic system in the brain. These findings suggest that 5-IT poses a substantial risk for abuse and addiction in humans.

Long lasting effects of chronic WIN55,212-2 treatment on mesostriatal dopaminergic and cannabinoid systems in the rat brain

Cannabinoid administration modulates dopamine transmission via an indirect, multisynaptic mechanism that includes the activation of cannabinoid type-1 receptor (CB1R). The present study evaluated in rodents, the effects of acute and chronic (20 days) WIN55,212-2 administration, a non-selective CB1R agonist, on dopamine uptake and synthesis in the mesolimbic and nigrostriatal dopaminergic pathways and associate them to its effects on the endocannabinoid system. The effect of spontaneous withdrawal, after different abstinence periods (7 days, 20 days), was also assessed. Acute and chronic administration of WIN55,212-2 decreased dopamine transporter (DAT) binding and mRNA levels, as well as tyrosine hydroxylase (TH) mRNA expression in the substantia nigra (SN) and ventral tegmental area (VTA). In the striatum, chronic WIN55,212-2 administration led to decreased protein expression of DAT and TH, whereas no alterations were observed after acute administration, suggesting a diminished dopamine uptake and synthesis after chronic agonist treatment. Furthermore, after chronic agonist treatment, we observed reduced CB1R binding and mRNA levels in SN and striatum, providing evidence for a possible regulatory role of the endocannabinoid system on dopaminergic function. Seven days after WIN55,212-2 cessation, we observed a rebound increase in mRNA, binding and total protein levels of DAT and TH in VTA, SN and striatum proposing the existence of a biphasic expression pattern, which was also observed in CB1R binding levels. Within the 20-day period of abstinence, TH mRNA and protein levels and CB1R binding levels remain increased. The above results indicate that chronic CB1R agonist treatment induces long-lasting control of the mesostriatal dopaminergic activity.

AZI23'UTR Is a New SLC6A3 Downregulator Associated with an Epistatic Protection Against Substance Use Disorders.

Regulated activity of SLC6A3, which encodes the human dopamine transporter (DAT), contributes to diseases such as substance abuse disorders (SUDs); however, the exact transcription mechanism remains poorly understood. Here, we used a common genetic variant of the gene, intron 1 DNP1B sequence, as bait to screen and clone a new transcriptional activity, AZI23'UTR, for SLC6A3. AZI23'UTR is a 3' untranslated region (3'UTR) of the human 5-Azacytidine Induced 2 gene (AZI2) but appeared to be transcribed independently of AZI2. Found to be present in both human cell nuclei and dopamine neurons, this RNA was shown to downregulate promoter activity through a variant-dependent mechanism in vitro. Both reduced RNA density ratio of AZI23'UTR/AZI2 and increased DAT mRNA levels were found in ethanol-naive alcohol-preferring rats. Secondary analysis of dbGaP GWAS datasets (Genome-Wide Association Studies based on the database of Genotypes and Phenotypes) revealed significant interactions between regions upstream of AZI23'UTR and SLC6A3 in SUDs. Jointly, our data suggest that AZI23'UTR confers variant-dependent transcriptional regulation of SLC6A3, a potential risk factor for SUDs.

Valproate increases dopamine transporter expression through histone acetylation and enhanced promoter binding of Nurr1

The dopamine transporter (DAT) is the key regulator of dopaminergic transmission and is a target of several xenobiotics, including pesticides and pharmacological agents. Previously, we identified a prominent role for histone deacetylases in the regulation of DAT expression. Here, we utilized a rat dopaminergic cell line (N27) to probe the responsiveness of DAT mRNA expression to inhibitors of histone acetylation. Inhibition of histone deacetylases (HDACs) by valproate, butyrate and Trichostatin A led to a 3–10-fold increase in DAT mRNA expression, a 50% increase in protein levels, which were accompanied by increased H3 acetylation levels. To confirm the mechanism of valproate-mediated increase in DAT mRNA, chromatin immunoprecipitation (ChIP) assays were used and demonstrated a significant increase in enrichment of acetylation of histone 3 on lysines 9 and 14 (H3K9/K14ac) in the DAT promoter. Expression of Nurr1 and Pitx3, key regulators of DAT expression, were increased following valproate treatment and Nurr1 binding was enriched in the DAT promoter. Together, these results indicate that histone acetylation and subsequent enhancement of transcription factor binding are plausible mechanisms for DAT regulation by valproate and, perhaps, by other xenobiotics.

Dopaminergic control of anxiety in young and aged zebrafish

Changes in the expression of the dopamine transporter (DAT), or the sensitivity of dopamine receptors, are associated with aging and substance abuse and may underlie some of the symptoms common to both conditions. In this study, we explored the role of the dopaminergic system in the anxiogenic effects of aging and acute cocaine exposure by comparing the behavioral phenotypes of wild type (WT) and DAT knockout zebrafish (DAT-KO) of different ages. To determine the involvement of specific dopamine receptors in anxiety states, antagonists to D1 (SCH23390) and D2/D3 (sulpiride) were employed. We established that DAT-KO results in a chronic anxiety-like state, seen as an increase in bottom-dwelling and thigmotaxis. Similar effects were produced by aging and acute cocaine administration, both leading to reduction in DAT mRNA abundance (qPCR). Inhibition of D1 activity counteracted the anxiety-like effects associated with DAT deficit, independent of its origin. Inhibition of D2/D3 receptors reduced anxiety in young DAT-KO, and enhanced the anxiogenic effects of cocaine in WT, but did not affect aged WT or DAT-KO fish. These findings provide new evidence that the dopaminergic system plays a critical role in anxiety-like states, and suggest that adult zebrafish provide a sensitive diurnal vertebrate model for elucidating the molecular mechanisms of anxiety and a platform for anxiolytic drug screens.

The legacy pesticide dieldrin acts as a teratogen and alters the expression of dopamine transporter and dopamine receptor 2a in zebrafish (Danio rerio) embryos

Dieldrin (DLD) is a lipophilic pesticide that shows environmental persistence. The objectives were to determine the effects of DLD on GABAergic and dopaminergic systems in developing zebrafish. Both chorionated and dechorionated embryos (~24h post-hatch) were exposed to a single concentration of DLD (0.347–3470μM) for 48h. Following exposure, a subset of larvae was placed into clean water for 6days (i.e. depuration phase). Chorionated embryos showed <15% mortality while dechorionated embryos showed higher mortality (>30%), suggesting that the chorion protected the embryos. Over a 6day depuration phase, there was a dose dependent effect observed in both the “dechorionated and chorionated embryo” treatments for larval mortality (>60%). At the end of depuration, there was no detectable change in neuro-morphological endpoints that included the ratio of notochord length to body length (%) and the ratio of head area to body area (%). However, DLD did induce cardiac edema, skeletal deformities, and tremors. GABA-related transcripts were not affected in abundance by DLD. Conversely, the relative mRNA levels of dopamine transporter (dat1) and dopamine receptor drd2a mRNA were decreased in dechorionated, but not chorionated, embryos. These data suggest that DLD can alter the expression of transcripts related to dopaminergic signaling. Lastly, GABAA receptor subunits gabrB1 and gabrB2, as well as dopamine receptors drd1 and drd2a, were inherently higher in abundance in dechorionated embryos compared to chorionated embryos. This is an important consideration when incorporating transcriptomics into embryo testing as expression levels can change with removal of the chorion prior to exposure.

Putative presynaptic dopamine dysregulation in schizophrenia is supported by molecular evidence from post-mortem human midbrain

The dopamine hypothesis of schizophrenia posits that increased subcortical dopamine underpins psychosis. In vivo imaging studies indicate an increased presynaptic dopamine synthesis capacity in striatal terminals and cell bodies in the midbrain in schizophrenia; however, measures of the dopamine-synthesising enzyme, tyrosine hydroxylase (TH), have not identified consistent changes. We hypothesise that dopamine dysregulation in schizophrenia could result from changes in expression of dopamine synthesis enzymes, receptors, transporters or catabolic enzymes. Gene expression of 12 dopamine-related molecules was examined in post-mortem midbrain (28 antipsychotic-treated schizophrenia cases/29 controls) using quantitative PCR. TH and the synaptic dopamine transporter (DAT) proteins were examined in post-mortem midbrain (26 antipsychotic-treated schizophrenia cases per 27 controls) using immunoblotting. TH and aromatic acid decarboxylase (AADC) mRNA and TH protein were unchanged in the midbrain in schizophrenia compared with controls. Dopamine receptor D2 short, vesicular monoamine transporter (VMAT2) and DAT mRNAs were significantly decreased in schizophrenia, with no change in DRD3 mRNA, DRD3nf mRNA and DAT protein between diagnostic groups. However, DAT protein was significantly increased in putatively treatment-resistant cases of schizophrenia compared to putatively treatment-responsive cases. Midbrain monoamine oxidase A (MAOA) mRNA was increased, whereas MAOB and catechol-O-methyl transferase mRNAs were unchanged in schizophrenia. We conclude that, whereas some mRNA changes are consistent with increased dopamine action (decreased DAT mRNA), others suggest reduced dopamine action (increased MAOA mRNA) in the midbrain in schizophrenia. Here, we identify a molecular signature of dopamine dysregulation in the midbrain in schizophrenia that mainly includes gene expression changes of molecules involved in dopamine synthesis and in regulating the time course of dopamine action.

Genetic Isolation of Hypothalamic Neurons that Regulate Context-Specific Male Social Behavior.

Nearly all animals engage in a complex assortment of social behaviors that are essential for the survival of the species. In mammals, these behaviors are regulated by sub-nuclei within the hypothalamus, but the specific cell types within these nuclei responsible for coordinating behavior in distinct contexts are only beginning to be resolved. Here, we identify a population of neurons in the ventral premammillary nucleus of the hypothalamus (PMV) that are strongly activated in male intruder mice in response to a larger resident male but that are not responsive to females. Using a combination of molecular and genetic approaches, we demonstrate that these PMV neurons regulate intruder-specific male social behavior and social novelty recognition in a manner dependent on synaptic release of the excitatory neurotransmitter glutamate. These data provide direct evidence for a unique population of neurons that regulate social behaviors in specific contexts.

Estrogen modulation of calretinin and BDNF expression in midbrain dopaminergic neurons of ovariectomised mice

Estrogen attenuates the loss of dopamine neurons from the substantia nigra in animal models of Parkinson’s disease (PD) and excitatory amino-acid induced neurotoxicity by interactions with brain-derived neurotrophic factor (BDNF), and calretinin (CR) containing dopaminergic (DA) neurons. To examine this interaction more closely, we treated the ovariectomised (OVX) mice with estrodial for 10days, and compared these mice to those OVX mice injected with the vehicle or control mice. Estrogen treatment in OVX mice had significantly more tyrosine hydroxylase (TH) positive neurons in the substantia nigra pars compacta (SNpc). Dopamine transporter (DAT) mRNA and BDNF mRNA levels in the midbrain were also significantly increased by estrogen treatment (P<0.05). OVX markedly decreased the number of TH/CR double stained cells in the SNpc (P<0.05), a trend which could be reversed by estrogen treatment. However, the number of GFAP positive cells in the substantia nigra did not show significant changes (P >0.05) after vehicle or estrodial treatment. Furthermore, we found that estrogen treatment abrogated the OVX-induced decrease in the phosphorylated AKT (p-AKT), but not p-ERK. We hypothesize that short-term treatment with estrogen confers neuroprotection to DA neurons by increasing CR in the DA neurons and BDNF in the midbrain, which possibly related to activation of the PI3K/Akt signaling pathway.

High sucrose consumption during pregnancy induced ADHD-like behavioral phenotypes in mice offspring

In recent years, the average consumption of sugar in humans from all ages has remarkably increased, exceeding the recommended limit. Pregnancy is a critical time for the global development of offsprings who are vulnerable to the deleterious effects of environmental factors. In this study, we investigated whether high sucrose consumption during pregnancy could affect the attention-deficit hyperactivity disorder (ADHD)-like neurobehavioral outcomes in offspring mice. Pregnant mice were randomly grouped and orally administered with either water as control (Con) or 30% wt/vol sucrose diluted in water at 6 (Suc6) or 9 (Suc9) g/kg dosage per day from gestational days 6 to 15. After the weaning period, offspring mice underwent a series of behavioral testing for locomotor activity, attention, and impulsivity. Although there is no obvious difference in gross development of offspring mice such as weight gain, high sucrose-exposed offspring mice showed a significantly increased locomotor activity. Moreover, these mice exhibited a dose-dependent decrease in attention and increase in impulsivity. In the striatum, a significantly increased dopamine transporter (DAT) mRNA expression was found in the Suc9 group along with dose-dependent decreases in the Drd1, Drd2 and Drd4 dopamine receptor subtypes. Furthermore, synaptosomal DAT protein expression was increased about twofold in the Suc9 group. Prenatal fructose exposure also induced hyperactive behavior in offspring mice suggesting the essential role of fructose in the dysregulated neurobehavioral development. These findings suggest prenatal sucrose consumption as a new risk factor for ADHD, which may need further attention and investigation in humans.

Relative Timing Between Kappa Opioid Receptor Activation and Cocaine Determines the Impact on Reward and Dopamine Release.

Negative affective states can increase the rewarding value of drugs of abuse and promote drug taking. Chronic cocaine exposure increases levels of the neuropeptide dynorphin, an endogenous ligand at kappa opioid receptors (KOR) that suppresses dopamine release in the nucleus accumbens (NAc) and elicits negative affective states upon drug withdrawal. However, there is evidence that the effects of KOR activation on affective state are biphasic: immediate aversive effects are followed by delayed increases in reward. The impact of KOR-induced affective states on reward-related effects of cocaine over time is not known. We hypothesize that the initial aversive effects of KOR activation increase, whereas the delayed rewarding effects decrease, the net effects of cocaine on reward and dopamine release. We treated rats with cocaine at various times (15 min to 48 h) after administration of the selective KOR agonist salvinorin A (salvA). Using intracranial self-stimulation and fast scan cyclic voltammetry, we found that cocaine-induced increases in brain stimulation reward and evoked dopamine release in the NAc core were potentiated when cocaine was administered within 1 h of salvA, but attenuated when administered 24 h after salvA. Quantitative real-time PCR was used to show that KOR and prodynorphin mRNA levels were decreased in the NAc, whereas tyrosine hydroxylase and dopamine transporter mRNA levels and tissue dopamine content were increased in the ventral tegmental area 24 h post-salvA. These findings raise the possibility that KOR activation-as occurs upon withdrawal from chronic cocaine-modulates vulnerability to cocaine in a time-dependent manner.

Epigenetic Regulation of Dopamine Transporter mRNA Expression in Human Neuroblastoma Cells.

The dopamine transporter (DAT) is a key regulator of dopaminergic neurotransmission. As such, proper regulation of DAT expression is important to maintain homeostasis, and disruption of DAT expression can lead to neurobehavioral dysfunction. Based on genomic features within the promoter of the DAT gene, there is potential for DAT expression to be regulated through epigenetic mechanisms, including DNA methylation and histone acetylation. However, the relative contribution of these mechanisms to DAT expression has not been empirically determined. Using pharmacologic and genetic approaches, we demonstrate that inhibition of DNA methyltransferase (DNMT) activity increased DAT mRNA approximately 1.5-2 fold. This effect was confirmed by siRNA knockdown of DNMT1. Likewise, the histone deacetylase (HDAC) inhibitors valproate and butyrate also increased DAT mRNA expression, but the response was much more robust with expression increasing over tenfold. Genetic knockdown of HDAC1 by siRNA also increased DAT expression, but not to the extent seen with pharmacological inhibition, suggesting additional isoforms of HDAC or other targets may contribute to the observed effect. Together, these data identify the relative contribution of DNMTs and HDACs in regulating expression. These finding may aid in understanding the mechanistic basis for changes in DAT expression in normal and pathophysiological states.

Striatal dopamine receptor plasticity in neurotensin deficient mice

Schizophrenia is thought to be caused, at least in part, by dysfunction in striatal dopamine neurotransmission. Both clinical studies and animal research have implicated the dopamine neuromodulator neurotensin (NT) in the pathophysiology of schizophrenia. Utilizing male mice lacking the NT gene (NT−/−), these studies examined the consequences of NT deficiency on dopaminergic tone and function, investigating (1) dopamine concentrations and dopamine receptor and transporter expression and binding in dopaminergic terminal regions, and (2) the behavioral effects of selective dopamine receptor agonists on locomotion and sensorimotor gating in adult NT−/− mice compared to wildtype (NT+/+) mice. NT−/− mice did not differ from NT+/+ mice in concentrations of dopamine or its metabolite DOPAC in any brain region examined. However, NT−/− mice showed significantly increased D1 receptor, D2 receptor, and dopamine transporter (DAT) mRNA in the caudate putamen compared to NT+/+ controls. NT−/− mice also showed elevated D2 receptor binding densities in both the caudate putamen and nucleus accumbens shell compared to NT+/+ mice. In addition, some of the behavioral effects of the D1-type receptor agonist SKF-82958 and the D2-type receptor agonist quinpirole on locomotion, startle amplitude, and prepulse inhibition were dose-dependently altered in NT−/− mice, showing altered D1-type and D2-type receptor sensitivity to stimulation by agonists in the absence of NT. The results indicate that NT deficiency alters striatal dopamine receptor expression, binding, and function. This suggests a critical role for the NT system in the maintenance of striatal DA system homeostasis and implicates NT deficiency in the etiology of dopamine-associated disorders such as schizophrenia.

Dopaminergic activity and behaviour in SOCS2 transgenic mice: Revealing a potential drug target for schizophrenia.

Alterations in immune function have been implicated in the aetiopathogenesis of schizophrenia. Specifically, the induction of inflammatory cytokines, which are important immunological factors in infection or inflammation, may be critical factors altering the normal course of brain development and increasing schizophrenia risk. Suppressor of cytokine signalling 2 (SOCS2) can negatively regulate the signalling of cytokines. The present study aimed to determine the behavioural phenotype of transgenic mice over-expressing SOCS2 (SOCS2 Tg) in paradigms of relevance to schizophrenia. Both male and female SOCS2 Tg mice displayed reduced locomotor hyperactivity after the administration of the dopamine releaser, amphetamine, compared to wildtype controls (WT). However, only male SOCS2 Tg mice showed enhanced prepulse inhibition compared to WT. Dopamine D2 receptors mRNA expression was reduced and dopamine transporter mRNA expression was increased in the nucleus accumbens of female, but not male, SOCS2 Tg mice, compared to WT. The role of hyperdopaminergia has long been implicated in the aetiology of schizophrenia. This study shows that over-expression of SOCS2 reduces the psychostimulant effects of amphetamine, enhances PPI, and alters mesolimbic dopaminergic activity. SOCS2 may provide a novel target in the development of treatments for schizophrenia.

N-terminal tagging of the dopamine transporter impairs protein expression and trafficking in vivo.

The dopamine transporter (DAT) is the primary protein responsible for the uptake of dopamine from the extracellular space back into presynaptic neurons. As such, it plays an important role in the cessation of dopaminergic neurotransmission and in the maintenance of extracellular dopamine homeostasis. Here, we report the development of a new BAC transgenic mouse line that expresses DAT with an N-terminal HA-epitope (HAD-Tg). In this line, two copies of the HA-DAT BAC are incorporated into the genome, increasing DAT mRNA levels by 47%. Despite the increase in mRNA levels, HAD-Tg mice show no significant increase in the level of DAT protein in the striatum, indicating a defect in protein trafficking or stability. By crossing HAD-Tg mice with DAT knockout mice (DAT-KO), we engineered mice that exclusively express HA-tagged DAT in the absence of endogenous DAT (DAT-KO/HAD-Tg). We show that DAT-KO/HAD-Tg mice express only 8.5% of WT DAT levels in the striatum. Importantly, the HA-tagged DAT that is present in DAT-KO/HAD-Tg mice is functional, as it is able to partially rescue the DAT-KO hyperactive phenotype. Finally, we provide evidence that the HA-tagged DAT is retained in the cell body based on a reduction in the striatum:midbrain protein ratio. These results demonstrate that the presence of the N-terminal tag leads to impaired DAT protein expression in vivo due in part to improper trafficking of the tagged transporter, and highlight the importance of the N-terminus in the transport of DAT to striatal terminals.

Testosterone induces molecular changes in dopamine signaling pathway molecules in the adolescent male rat nigrostriatal pathway.

Adolescent males have an increased risk of developing schizophrenia, implicating testosterone in the precipitation of dopamine-related psychopathology. Evidence from adult rodent brain indicates that testosterone can modulate nigrostriatal dopamine. However, studies are required to understand the role testosterone plays in maturation of dopamine pathways during adolescence and to elucidate the molecular mechanism(s) by which testosterone exerts its effects. We hypothesized that molecular indices of dopamine neurotransmission [synthesis (tyrosine hydroxylase), breakdown (catechol-O-methyl transferase; monoamine oxygenase), transport [vesicular monoamine transporter (VMAT), dopamine transporter (DAT)] and receptors (DRD1-D5)] would be changed by testosterone or its metabolites, dihydrotestosterone and 17β-estradiol, in the nigrostriatal pathway of adolescent male rats. We found that testosterone and dihydrotestosterone increased DAT and VMAT mRNAs in the substantia nigra and that testosterone increased DAT protein at the region of the cell bodies, but not in target regions in the striatum. Dopamine receptor D2 mRNA was increased and D3 mRNA was decreased in substantia nigra and/or striatum by androgens. These data suggest that increased testosterone at adolescence may change dopamine responsivity of the nigrostriatal pathway by modulating, at a molecular level, the capacity of neurons to transport and respond to dopamine. Further, dopamine turnover was increased in the dorsal striatum following gonadectomy and this was prevented by testosterone replacement. Gene expression changes in the dopaminergic cell body region may serve to modulate both dendritic dopamine feedback inhibition and reuptake in the dopaminergic somatodendritic field as well as dopamine release and re-uptake dynamics at the presynaptic terminals in the striatum. These testosterone-induced changes of molecular indices of dopamine neurotransmission in males are primarily androgen receptor-driven events as estradiol had minimal effect. We conclude that nigrostriatal responsivity to dopamine may be modulated by testosterone acting via androgen receptors to alter gene expression of molecules involved in dopamine signaling during adolescence.