Anderson Manoel Herculano

Measuring anxiety in zebrafish: A critical review

Zebrafish are increasingly being used in behavioral neuroscience, neuropsychopharmacology and neurotoxicology. Recently, behavioral screens used to model anxiety in rodents were adapted to this species, and novel models which tap on zebrafish behavioral ecology have emerged. However, model building is an arduous task in experimental psychopathology, and a continuous effort to assess the validity of these measurements is being chased among some researchers. To consider a model as valid, it must possess face, predictive and/or construct validity. In this article, we first review some notions of validity, arguing that, at its limit, face and predictive validity reduce to construct validity. Then we review some procedures which have been used to study anxiety, fear or related processes in zebrafish, using the validity framework. We conclude that, although the predictive validity of some of these models is increasingly being met, there is still a long way in reaching the desired level of construct validity. The refinement of models is an ongoing activity, and behavioral validation and parametric research ought to advance that objective.

Mercury and human genotoxicity: Critical considerations and possible molecular mechanisms

Mercury compounds versatility explains their numerous applications in diverse areas of industry. The growing use of this metal has resulted in a significant increase of environment contamination and episodes of human intoxication, arousing the concern of international organisms. Meanwhile, consequences of these intoxication outbreaks are still not fully understood, especially if we consider long-term effects of chronic exposure to relatively low levels of mercury compounds. In the present manuscript, studies about the genotoxicity of mercury compounds, performed in vitro, in vivo, and/or including epidemiologic studies of human populations were reviewed. Some mercury compounds are known as teratogenic agents, especially affecting the normal development of the central nervous system; however, the connection between mercury exposure and carcinogenesis remains controversial. Since 1990s, epidemiological studies have begun to include an increasing number of human subjects, making the results more reliable: thus, increased genotoxicity was demonstrated in human populations exposed to mercury through diet, occupation or by carrying dental fillings. In fact, concentrations of methylmercury causing significant genotoxic alterations in vitro below both safety limit and concentration were associated with delayed psychomotor development with minimal signs of methylmercury poisoning. Based on mercurys known ability to bind sulfhydryl groups, several hypotheses were raised about potential molecular mechanisms for the metal genotoxicity. Mercury may be involved in four main processes that lead to genotoxicity: generation of free radicals and oxidative stress, action on microtubules, influence on DNA repair mechanisms and direct interaction with DNA molecules. All data reviewed here contributed to a better knowledge of the widespread concern about the safety limits of mercury exposure.

Pharmacological analysis of zebrafish (Danio rerio) scototaxis

The scototaxis test has been introduced recently to assess anxiety-like phenotypes in fish, including zebrafish. Parametric analyses suggest that scototaxis represents an approach-avoidance conflict, which hints at anxiety. In this model, white avoidance represents anxiety-like behavior, while the number of shuttling events represents activity. Acute or chronic fluoxetine, buspirone, benzodiazepines, ethanol, caffeine and dizocilpine were assessed using the light-dark box (scototaxis) test in zebrafish. Acute fluoxetine treatment did not alter white avoidance, but altered locomotion in the higher dose; chronic treatment (2 weeks), on the other hand, produced an anxiolytic effect with no locomotor outcomes. The benzodiazepines produced a hormetic (inverted U-shaped) dose-response profile, with intermediate doses producing anxiolysis and no effect at higher doses; clonazepam, a high-potency benzodiazepine agonist, produced a locomotor impairment at the highest dose. Buspirone produced an anxiolytic profile, without locomotor impairments. Moclobemide did not produce behavioral effects. Ethanol also produced a hormetic profile in white avoidance, with locomotor activation in 0.5% concentration. Caffeine produced an anxiogenic profile, without locomotor effects. These results suggest that the light-dark box is sensitive to anxiolytic and anxiogenic drugs in zebrafish.

Mercury in the Tapajós River basin, Brazilian Amazon: A review

This paper presents a review about mercury contamination and human exposure in the Tapajós River basin (Brazil), one of the major tributaries of the Amazon impacted by traditional gold mining from the mid 1980s. The most recent review in this region was published more than ten years ago and since then many articles about environment and especially human populations have revealed new aspects of mercury toxicology. Additionally, new biomarkers of mercury exposure and toxicity have been studied in these populations. However, there are still many open, about both mercurys biogeochemical cycle and mercury health risks. Further environmental and human risk research directions are proposed.

Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models.

Serotonin (5-HT) is a neurotransmitter that is involved in many behavioral functions, including the organization of defense, and its putative pathological correlate, anxiety and stress disorders. Recently, behavioral tests for anxiety have been proposed in zebrafish. Exposure to the novel tank test or to the light/dark test increased extracellular fluid 5-HT content in the brain; anxiety-like behavior correlated positively with 5-HT content in the novel tank test, while the correlation was negative in the light/dark test. Acute treatment with a low dose of fluoxetine was anxiolytic in the geotaxis test and anxiogenic in the scototaxis test, while treatment with a higher dose produced a hyperlocomotor effect in both tasks. Buspirone and WAY 100635 were anxiolytic in both tests, while SB 224289 was anxiolytic in the geotaxis and slightly anxiogenic in the scototaxis test. Serotonin depletion with pCPA was anxiogenic in the geotaxis and anxiolytic in scototaxis. These results underline the differential sensitivity of these tasks to assess serotonergic agents; alternatively, serotonin might regulate zebrafish behavior differently in the novel tank test and in the light/dark test.

A Review of Monoaminergic Neuropsychopharmacology in Zebrafish

Monoamine neurotransmitters are the major regulatory mechanisms in the vertebrate brain, involved in the adjustment of motivation, emotion, and cognition. The chemical anatomy of these systems is thought to be highly conserved in the brain of all vertebrates, including zebrafish. Recently, the development of behavioral assays in zebrafish allowed the neuropsychopharmacological investigation of these circuits and its functions. Here we review neuroanatomical, genetic, neurochemical, and psychopharmacological evidence regarding the roles of histaminergic, dopaminergic, noradrenergic, serotonergic, and melatonergic systems in this species. We conclude that, in spite of species differences, zebrafish are suitable for the investigation of neuropsychopharmacology of drugs that affect theses systems; nonetheless, more thorough validation of behavioral methods is still needed.

Serotonergic modulation of zebrafish behavior: towards a paradox.

Due to the fish-specific genome duplication event (~320-350 mya), some genes which code for serotonin proteins were duplicated in teleosts; this duplication event was preceded by a reorganization of the serotonergic system, with the appearance of the raphe nuclei (dependent on the isthmus organizer) and prosencephalic nuclei, including the paraventricular and pretectal complexes. With the appearance of amniotes, duplicated genes were lost, and the serotonergic system was reduced to a more complex raphe system. From a comparative point of view, then, the serotonergic system of zebrafish and that of mammals shows many important differences. However, many different behavioral functions of serotonin, as well as the effects of drugs which affect the serotonergic system, seem to be conserved among species. For example, in both zebrafish and rodents acute serotonin reuptake inhibitors (SSRIs) seem to increase anxiety-like behavior, while chronic SSRIs decrease it; drugs which act at the 5-HT1A receptor seem to decrease anxiety-like behavior in both zebrafish and rodents. In this article, we will expose this paradox, reviewing the chemical neuroanatomy of the zebrafish serotonergic system, followed by an analysis of the role of serotonin in zebrafish fear/anxiety, stress, aggression and the effects of psychedelic drugs.

Adenosine A1, but not A2, Receptor Blockade Increases Anxiety and Arousal in Zebrafish

Adenosinergic systems have been implicated in anxiety-like states, as caffeine can induce a state of anxiety in human beings. Caffeine is an antagonist at A(1) and A(2) adenosine receptors but it remains unclear whether anxiety is mediated by one or both of these. As the adenosinergic system is rather conserved, we opted to pursue these questions using zebrafish, a widely used model organism in genetics and developmental biology. Zebrafish adenosine 1. 2A.1 and 2A.2 receptors conserve histidine residues in TM6 and TM7 that are responsible for affinity in bovine A1 receptor. We investigated the effects of caffeine, PACPX (an A(1) receptor antagonist) and 1,3-dimethyl-1-propargylxanthine (DMPX) (an A(2) receptor antagonist) on anxiety-like behaviour and locomotor activity of zebrafish in the scototaxis test as well as evaluated the effects of these drugs on pigment aggregation. Caffeine increased anxiety at the dose of 100 mg/kg, while locomotion at the dose of 10 mg/kg was increased. Both doses of 10 and 100 mg/kg induced pigment aggregation. PACPX, on the other hand, increased anxiety at a dose of 6 mg/kg and induced pigment aggregation at the doses of 0.6 and 6 mg/kg, but did not produce a locomotor effect. DMPX, in turn, increased locomotion at the dose of 6 mg/kg but did not produce any effect on pigment aggregation or anxiety-like behaviour. These results indicate that blockade of A(1)-R, but not A(2)-R, induces anxiety and autonomic arousal, while the blockade of A(2)-R induces hyperlocomotion. Thus, as in rodents, caffeines anxiogenic and arousing effects are probably mediated by A(1) receptors in zebrafish and its locomotor activating effect is probably mediated by A(2) receptors.

Behavioral and neurochemical changes in the zebrafish leopard strain

The zebrafish leopard phenotype (leo) displays abnormal pigmentation and shows increased anxiety‐like behavior. The neurochemical changes associated with this anxious phenotype are not known. Here, we demonstrate that leo show increased anxiety‐like behavior in the light/dark box and in the novel tank test. This anxious phenotype is rescued by acute treatment with a dose of a serotonin reuptake inhibitor, fluoxetine, that is inactive in wild‐type animals. Moreover, leo show decreased tissue levels of serotonin, increased serotonin turnover and slightly increased monoamine oxidase activity. These results suggest that the anxious phenotype observed in leo zebrafish is caused by a decrease in serotonin uptake. This work could open an important avenue in defining the neurochemical underpinning of natural variation in anxiety disorders.

Possible role of serotoninergic system in the neurobehavioral impairment induced by acute methylmercury exposure in zebrafish (Danio rerio)

Adult zebrafish were treated acutely with methylmercury (1.0 or 5.0 μg g(-1), i.p.) and, 24h after treatment, were tested in two behavioral models of anxiety, the novel tank and the light/dark preference tests. At the smaller dose, methylmercury produced a marked anxiogenic profile in both tests, while the greater dose produced hyperlocomotion in the novel tank test. These effects were accompanied by a decrease in extracellular levels of serotonin, and an increase in extracellular levels of tryptamine-4,5-dione, a partially oxidized metabolite of serotonin. A marked increase in the formation of malondialdehyde, a marker of oxidative stress, accompanied these parameters. It is suggested that methylmercury-induced oxidative stress produced mitochondrial dysfunction and originated tryptamine-4,5-dione, which could have further inhibited tryptophan hydroxylase. These results underscore the importance of assessing acute, low-level neurobehavioral effects of methylmercury.