Magda Nowicki

Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner

Motor function and anxiety-like responses are easily quantifiable in zebrafish, a novel model organism for behavioral pharmacology. Activation of serotonin receptors through the use of selective agonists has been shown to alter anxiety-like behaviors in zebrafish. However, few studies have examined the effect of blockade of specific serotonin receptors. In the current study, we examine the effect of 4 serotonin receptor antagonists selective for 5-HT1A, 5-HT1B/D, 5-HT2, and 5-HT3 receptors on zebrafish motor and anxiety-like responses. Exposure to the receptor antagonists did not change baseline motor responses. However, when placed in a novel environment, zebrafish previously exposed to GR 55562 (5-HT1B/D antagonist) exhibited reduced anxiety-like behavior, whereas zebrafish previously exposed to p-MPPF (5-HT1A antagonist), Ketanserin (5-HT2 antagonist), or Ondasetron (5-HT3 antagonist) exhibited increased anxiety-like behaviors. These results show that drugs developed for mammalian serotonin receptors are efficacious in the zebrafish too, a finding that demonstrates evolutionary conservation of the serotoninergic system. The results also imply that zebrafish may be an appropriate animal model for examining the serotonergic neurotransmitter system in vertebrates.

Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver

Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish.

Neurochemical factors underlying individual differences in locomotor activity and anxiety-like behavioral responses in zebrafish.

Variation among individuals may arise for several reasons, and may have diverse underlying mechanisms. Individual differences have been studied in a variety of species, but recently a new model organism has emerged in this field that offers both sophistication in phenotypical characterization and powerful mechanistic analysis. Recently, zebrafish, one of the favorites of geneticists, have been shown to exhibit consistent individual differences in baseline locomotor activity. In the current study, we further explore this finding and examine whether individual differences in locomotor activity correlate with anxiety-like behavioral measures and with levels of dopamine, serotonin and the metabolites of these neurotransmitters. In addition, we examine whether individual differences in locomotor activity are also associated with reactivity to the locomotor stimulant effects of and neurochemical responses to acute ethanol exposure (30min long, 1% v/v ethanol bath application). Principal component analyses revealed a strong association among anxiety-like responses, locomotor activity, serotonin and dopamine levels. Furthermore, ethanol exposure was found to abolish the locomotion-dependent anxiety-like behavioral and serotonergic responses suggesting that this drug also engages a common underlying pathway. Overall, our results provide support for an important role of the serotonergic system in mediating individual differences in anxiety-like responses and locomotor activity in zebrafish and for a minor modulatory role of the dopaminergic system.

Time-dependent interacting effects of caffeine, diazepam, and ethanol on zebrafish behaviour

ABSTRACT Zebrafish have become a popular animal model for behavioural pharmacology due to their small size, rapid development, and amenability to high throughput behavioural drug screens. Furthermore, water‐soluble compounds can be administered via immersion of the fish in the drug solution, which provides a non‐invasive drug delivery method. Numerous studies have demonstrated stimulant effects of alcohol. Diazepam and caffeine, on the other hand have been found to have inhibitory effect on locomotor activity in zebrafish. However, the time‐dependent changes induced by these psychoactive drugs are rarely reported, and potential drug interactions have not been examined in zebrafish, despite the translational relevance of this question. In the current study, we examine time‐ and dose‐dependent changes in zebrafish following exposure to caffeine, diazepam, and ethanol quantifying four different behavioural parameters over a 30 min recording session. We subsequently analyze potential drug‐drug interactions by co‐administering the three drugs in different combinations. Our time‐course and dose‐response analyses for each of the three drugs represent so far the most detailed studies available serving as a foundation for future psychopharmacology experiments with zebrafish. Furthermore, we report significant interactions between the three drugs corroborating findings obtained with rodent models as well as in humans, providing translational relevance for the zebrafish model. HIGHLIGHTSTime and dose‐dependent effects of caffeine, diazepam, and ethanol are reported.Significant interaction effects between caffeine and alcohol are reported.Significant interaction effects between alcohol and diazepam are reported.

Interaction between handling induced stress and anxiolytic effects of ethanol in zebrafish: A behavioral and neurochemical analysis

Stress is often considered an important factor in the development of alcohol addiction. In rodents, various types of stressors have been shown to potentiate the effects of alcohol on behavioral responses, and to increase consumption of this substance. However, few have investigated the interaction between stress and alcohol in zebrafish. In the current study we present a repeated handling stress paradigm we developed for zebrafish, and examine whether stress alters alcohol induced behavioral and neurochemical responses. Our results show that repeated handling of zebrafish conducted for 2 consecutive days is sufficient to increase anxiety-like behavioral responses quantified 24h post-stressor. Repeatedly handled zebrafish also exhibited a reduction in the levels of serotonins metabolite, 5-hydroxyindole acetic acid (quantified by high precision liquid chromatography) compared to unhandled controls. A 60-min acute exposure to 1% ethanol was found to significantly increase locomotor activity and decrease anxiety-like behavioral responses in stressed zebrafish but not in controls. Furthermore, unhandled control zebrafish exhibited a significant increase in whole-brain dopamine levels following exposure to ethanol but the increase was not observed in repeatedly handled fish. Our findings suggest that ethanol induced locomotor activity and anxiolysis is potentiated by handling stress and may be partially mediated by changes in dopaminergic and serotonergic activity. Overall, we demonstrate the validity of our repeated handling stressor paradigm for zebrafish, which can be used to investigate the interaction between stress and ethanol.

Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio).

Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamines metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohols locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

Acute alcohol exposure increases tyrosine hydroxylase protein expression and dopamine synthesis in zebrafish

Zebrafish have become a popular animal model for investigating the effects of alcohol on the brain and behaviour. Acute exposure to alcohol has been shown to alter dopaminergic signalling in zebrafish, but the underlying mechanisms have not been well defined. In the current study, we characterize the effects of alcohol on the zebrafish dopaminergic system by focusing on tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Using western blot analysis, we demonstrate that a 60min exposure to 1% alcohol increases tyrosine hydroxylase protein expression in the zebrafish brain. Enzymatic activity assays confirmed that alcohol also increases tyrosine hydroxylase enzymatic activity, whereas HPLC analysis demonstrated increased levels of whole-brain dopamine and its metabolite DOPAC. In addition to activation of the dopaminergic system, behavioural analysis revealed accompanying increase of distance traveled following 1% alcohol exposure. These findings suggest that acute alcohol exposure elevates dopamine synthesis via increased tyrosine hydroxylase protein expression. Our results support the hypothesis that alcohol alters dopaminergic signalling in the zebrafish brain in a similar manner as compared to mammals.

Ethanol-Induced ADH Activity in Zebrafish: Differential Concentration-Dependent Effects on High- Versus Low-Affinity ADH Enzymes.

Zebrafish express enzymes that metabolize ethanol in a manner comparable to that of mammals, including humans. We previously demonstrated that acute ethanol exposure increases alcohol dehydrogenase (ADH) activity in an inverted U-shaped dose-dependent manner. It was hypothesized that the biphasic dose-response was due to the increased activity of a high-affinity ADH isoform following exposure to low concentrations of ethanol and increased activity of a low-affinity ADH isoform following exposure to higher concentrations of ethanol. To test this hypothesis, we exposed zebrafish to different concentrations of ethanol (0%, 0.25%, 0.5%, and 1.0% v/v) for 30 min and measured the total ADH activity in the zebrafish liver. However, we also repeated this enzyme activity assay using a low concentration of the substrate (ethanol) to determine the activity of high-affinity ADH isoforms. We found that total ADH activity in response to ethanol induces an inverted U-shaped dose-response similar to our previous study. Using a lower substrate level in our enzyme assay targeting high-affinity isozymes, we found a similar dose-response. However, the difference in activity between the high and low substrate assays (high substrate activity - low substrate activity), which provide an index of activity for low-affinity ADH isoforms, revealed no significant effect of ethanol exposure. Our results suggest that the inverted U-shaped dose-response for total ADH activity in response to ethanol is driven primarily by high-affinity isoforms of ADH.