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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.

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Recent studies suggest that impaired glutathione synthesis and distorted dopaminergic transmission are important factors in the pathophysiology of schizophrenia. In the present study, on the postnatal days p5-p16, male pups were treated with the inhibitor of glutathione synthesis, L-buthionine-(S,R)- sulfoximine (BSO, 3.8 or 7.6 mmol/kg), and the dopamine uptake inhibitor, GBR 12,909 (5 mg/kg) alone or in combination, and prepulse inhibition of the acoustic startle response (PPI) was evaluated in adult 90-day-old rats. Moreover, the monoamine levels in the cortex and hippocampus of 16-day-old rats or 91-day-old rats were measured. The present results showed that administration of BSO at 3.8 mmol/kg led to a decreasing tendency in PPI for all tested prepulse intensities. In contrast, a combined treatment with BSO in both studied doses and GBR 12,909 did not induce significant deficits in PPI. Moreover, the results of biochemical studies indicated that treatment with BSO or GBR 12,909 alone induced a weak increase in the activity of dopaminergic, serotonergic, and noradrenergic systems in the frontal cortex and hippocampus of 16-day-old rats and 91-day-old rats. However, the combined administration of both substances allowed for maintaining the normal activity of monoaminergic systems in the rat brain. The most significant changes in the functioning of monoaminergic systems were observed in the frontal cortex of 16-day-old rats. Therefore, it seems that the frontal cortex of rat puppies is most sensitive to glutathione deficiencies resulting in increased oxidative stress in neurons. As a result, it can lead to cognitive and memory impairment.

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This is the first study aiming to develop a method for the long-term visualization of living nigrostriatal dopaminergic neurons using 1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine-BODIPY (GBR-BP), the original fluorescent substance, which is a derivative of GBR-12909, a dopamine uptake inhibitor. This method is based on the authors' hypothesis about the possibility of specifically internalizing into dopaminergic neurons substances with a high affinity for the dopamine transporter (DAT). Using a culture of mouse embryonic mesencephalic and LUHMES cells (human embryonic mesencephalic cells), as well as slices of the substantia nigra of adult mice, we have obtained evidence that GBR-BP is internalized specifically into dopaminergic neurons in association with DAT via a clathrin-dependent mechanism. Moreover, GBR-BP has been proven to be nontoxic. As we have shown in a primary culture of mouse metencephalon, GBR-BP is also specifically internalized into some noradrenergic and serotonergic neurons, but is not delivered to nonmonoaminergic neurons. Our data hold great promise for visualization of dopaminergic neurons in a mixed cell population to study their functioning, and can also be considered a new approach for the development of targeted drug delivery to dopaminergic neurons in pathology, including Parkinson's disease.

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Growing evidence suggests a role for brain-gut-microbiota axis in affective disorders including major depression and bipolar disorder (BD). Herein, we aim to explore, by employing germ-free (GF) mice, the effect of the indigenous microbiota in the development of mania-like behavior. Conventional and GF mice were evaluated for the hyperlocomotion induced by the dopamine transporter inhibitor GBR12909 (15 mg/Kg), a validated model for mania-like behavior. Inflammatory mediators and neurotrophic factors were quantified in the prefrontal cortex, hippocampus and striatum. Mice lacking indigenous microbiota were less susceptible to the mania-like behavior induced by GBR12909. This effect was associated with decreased levels of inflammatory cytokines such as IL-6 and TNF-α, along with increased concentrations of anti- inflammatory cytokines (IL-10) and of neurotrophins (BDNF and NGF). We provided the first evidence that gut-microbiota-brain axis participates in the development of mania-like behavior in rodents, possibly through neuroimmunepathways.

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Inhibition of dopamine transporter (DAT) by GBR12909 has been proposed as a pharmacological model of mania related to bipolar disorder (BD). Here we tested the hypothesis that GBR12909 injection impairs habituation and induces hyperlocomotion in mice, along with changes in cytokines and neurotrophic factors levels, as observed in BD patients. We also tested if lithium carbonate, sodium valproate and aripiprazole prevent GBR12909-induced locomotion. Male Swiss mice received GBR12909 (15 mg/kg) injections and locomotor responses were quantified in an open field. Cytokines and neurotrophic factors levels were assessed in the prefrontal cortex, striatum and hippocampus 30 min and 24 h after injections. Pre-treatments with lithium, valproate or aripiprazole were performed with single and repeated injection protocols. GBR12909 prevented motoric habituation and increased basal locomotion in habituated mice in the open field. This compound also induced changes in IL-2 and BDNF levels in prefrontal cortex; IL-2, IL-4 and IL-10 in striatum; and IL-10, IL-4, IFN-γ and NGF in hippocampus. GBR12909-induced hyperlocomotion was attenuated by lithium (12.5-100 mg/kg), but not valproate (75-300 mg/kg), and prevented by aripiprazole (0.1-10 mg/kg). Repeated injections of these drugs (twice a day for 3 days), however, failed to inhibit hyperlocomotion. The main limitations of the protocols in this study are the analysis of locomotion as the only behavioral parameter, changes in immune factors that may overlap with other psychiatric disorders and the lack chronic drug injections. Despite of these limitations, this study adds to previous literature suggesting DAT inhibition as a potential animal model of mania related to BD.

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BACKGROUND: Mania/hypomania is the cardinal feature of bipolar disorder. Recently, single administration of the dopamine transporter (DAT) inhibitor, GBR12909, was related to mania-like alterations. In the present study we aimed at testing behavioral and brain oxidant/neurotrophic alterations induced by the repeated administration of GBR12909 and its prevention/reversal by the mood stabilizing drugs, lithium (Li) and valproate (VAL) as well as by the neuroprotective drug, minocycline (Mino). METHODS: Adult Swiss mice were submitted to 14 days protocols namely prevention and reversal. In the reversal protocol mice were given GBR12909 or saline and between days 8 and 14 received Li, VAL, Mino (25 or 50mg/kg) or saline. In the prevention treatment, mice were pretreated with Li, VAL, Mino or saline prior to GBR12909. RESULTS: GBR12909 repeated administration induced hyperlocomotion and increased risk taking behavior that were prevented and reversed by the mood stabilizers and both doses of Mino. Li, VAL or Mino were more effective in the reversal of striatal GSH alterations induced by GBR12909. Regarding lipid peroxidation Mino was more effective in the prevention and reversal of lipid peroxidation in the hippocampus whereas Li and VAL prevented this alteration in the striatum and PFC. Li, VAL and Mino25 reversed the decrease in BDNF levels induced by GBR12909. CONCLUSION: GBR12909 repeated administration resembles manic phenotype. Similarly to classical mood-stabilizing agents, Mino prevented and reversed GBR12909 manic-like behavior in mice. Thus, our data provide preclinical support to the design of trials investigating Mino's possible antimanic effects.

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The adolescent period in mammals is a critical period of brain maturation and thus represents a time of susceptibility to environmental insult, e.g. psychosocial stress and/or drugs of abuse, which may cause lasting impairments in brain function and behavior and even precipitate symptoms in at-risk individuals. One likely effect of these environmental insults is to increase oxidative stress in the developing adolescent brain. Indeed, there is increasing evidence that redox dysregulation plays an important role in the development of schizophrenia and other neuropsychiatric disorders and that GABA interneurons are particularly susceptible to alterations in oxidative stress. The current study sought to model this adolescent neurochemical "stress" by exposing mice to the dopamine transporter inhibitor GBR12909 (5mg/kg; IP) during adolescence (postnatal day 35-44) and measuring the resultant effect on locomotor behavior and probabilistic reversal learning as well as GABAergic interneurons and oxidative stress in adulthood. C57BL6/J mice exposed to GBR12909 showed increased activity in a novel environment and increased impulsivity as measured by premature responding in the probabilistic reversal learning task. Adolescent GBR12909-exposed mice also showed decreased parvalbumin (PV) immunoreactivity in the prefrontal cortex, which was accompanied by increased oxidative stress in PV+ neurons. These findings indicate that adolescent exposure to a dopamine transporter inhibitor results in loss of PV in GABAergic interneurons, elevations in markers of oxidative stress, and alterations in behavior in adulthood.

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BACKGROUND: Bipolar disorder (BD) mania patients exhibit poor cognition and reward-seeking/hypermotivation, negatively impacting a patient's quality of life. Current treatments (e.g., lithium), do not treat such deficits. Treatment development has been limited due to a poor understanding of the neural mechanisms underlying these behaviors. Here, we investigated putative mechanisms underlying cognition and reward-seeking/motivational changes relevant to BD mania patients using two validated mouse models and neurochemical analyses. METHODS: The effects of reducing dopamine transporter (DAT) functioning via genetic (knockdown vs. wild-type littermates), or pharmacological (GBR12909- vs. vehicle-treated C57BL/6J mice) means were assessed in the probabilistic reversal learning task (PRLT), and progressive ratio breakpoint (PRB) test, during either water or chronic lithium treatment. These tasks quantify reward learning and effortful motivation, respectively. Neurochemistry was performed on brain samples of DAT mutants ± chronic lithium using high performance liquid chromatography. RESULTS: Reduced DAT functioning increased reversals in the PRLT, an effect partially attenuated by chronic lithium. Chronic lithium alone slowed PRLT acquisition. Reduced DAT functioning increased motivation (PRB), an effect attenuated by lithium in GBR12909-treated mice. Neurochemical analyses revealed that DAT knockdown mice exhibited elevated homovanillic acid levels, but that lithium had no effect on these elevated levels. CONCLUSIONS: Reducing DAT functioning recreates many aspects of BD mania including hypermotivation and improved reversal learning (switching), as well as elevated homovanillic acid levels. Chronic lithium only exerted main effects, impairing learning and elevating norepinephrine and serotonin levels of mice, not specifically treating the underlying mechanisms identified in these models.

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RATIONALE: Modafinil is a wake-promoting drug with FDA approval for the treatment of excessive daytime sleepiness that has been prescribed for ADHD and recently assessed as a potential treatment for psychostimulant dependence. Previous research indicates that modafinil modestly increases locomotor activity and produces similar discriminative stimulus effects to psychostimulants in rodents, although the subjective effects of modafinil are reportedly distinct from those of cocaine or amphetamine in humans with a history of psychostimulant abuse. OBJECTIVES: The current study employed drug discrimination procedures in rats to examine the pharmacological actions contributing to modafinil's discriminative stimulus functions. METHODS: Eight male Sprague-Dawley rats were trained to discriminate intragastric administration of 256 mg/kg modafinil from vehicle (5% arabic gum) under a FR 20 schedule of food reinforcement. Substitution tests were conducted with various dopaminergic agents (d-amphetamine, cocaine, PNU-91356A, GBR 12909, methylphenidate) and nondopaminergic agents (nicotine, ethanol). Antagonist tests were conducted with the selective D1 antagonist, SCH 39166, and the nonselective D2 antagonist, haloperidol. RESULTS: Rats trained to discriminate modafinil displayed complete stimulus generalization to cocaine, methylphenidate, and GBR 12909 and the discrimination was completely blocked by both SCH 39166 and haloperidol. Evidence for significant partial substitution was obtained with d-amphetamine, PNU-91356A, and nicotine. CONCLUSIONS: Results strongly support the role of dopaminergic mechanisms in the discriminative stimulus functions of modafinil, although further evaluation regarding the contribution of other neurotransmitter systems to these effects should be continued. The findings are discussed in light of clinical research efforts with modafinil as a treatment for psychostimulant dependence.

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Pharmacological magnetic resonance imaging (phMRI) is a powerful tool for imaging the effects of drugs on brain activity. In preclinical phMRI studies, general anesthesia used for minimizing head movements is thought to influence the phMRI responses to drugs. In this study we investigated the phMRI responses to a selective dopamine transporter (DAT) inhibitor, GBR12909, and a dopamine (DA) releaser, d-amphetamine (AMPH), in the isoflurane anesthetized and awake rats using a relative cerebral blood volume (rCBV) method. AMPH (1 mg/kg i.p.) caused an increase in rCBV in the dopaminergic circuitry in the both anesthetized and awake rats. The striatal rCBV change was correlated with the change of the striatal DA concentration induced by AMPH in the both anesthetized and awake rats. GBR12909 (10 mg/kg i.p.) caused a positive rCBV response and showed a similar regional pattern of rCBV response to AMPH in the awake rats, and the correlation between the change of the striatal rCBV and the striatal DA concentration was observed. However, in the anesthetized rats, GBR12909 induced a widespread negative rCBV response, whereas an increase in striatal DA concentration was observed. These findings indicate that phMRI responses to activation of DA neurotransmission by GBR12909 or AMPH are overall identical in the awake state, while the phMRI response to a DAT inhibitor, GBR12909 but not to AMPH was changed by isoflurane anesthesia. For the evaluation of neuroactive drugs using phMRI, isoflurane anesthesia might be complicated the interpretation of pharmacodynamic effects of drugs in preclinical studies.

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