Vanessa A. Quadros

Strain- and context-dependent behavioural responses of acute alarm substance exposure in zebrafish.

We investigate the behavioural responses of wild type (WT) and leopard (leo) zebrafish elicited by alarm substances of conspecifics at three contexts: during the exposure period (Experiment 1); after exposure, in habituation to novelty (Experiment 2); or after exposure, in the light-dark preference test (Experiment 3), and analyse their influence on pigment response. During the exposure, leo showed decreased vertical drifts, increased number and duration of erratic movements, while WT had increased erratic movements and latency to enter the top. In the novel tank, we observed that angular velocity decreased in WT exposed to alarm substance, which also presented increased fear responses. Contrastingly, leo increased the number of entries and time in top, indicating differences in habituation profile. Alarm substance increased the number of erratic movements in the light-dark test, but elicited different responses between strains in scototaxis, latency to enter the dark compartment and risk assessment episodes. Moreover, the body colour of zebrafish did not change after alarm substance exposure. Principal component analyses suggest that burst swimming, anxiety-like behaviours, and locomotion/exploration were the components that most accounted for total variances of Experiments 1, 2, and 3, respectively. We conclude that chemical cue from conspecifics triggers strain- and context-dependent responses.

Modulatory action of taurine on ethanol-induced aggressive behavior in zebrafish.

Alcohol is a potent agent for eliciting aggression in vertebrates. Taurine (TAU) is an amino sulfonic acid with pleiotropic actions on brain function. It is one of the most abundant molecules present in energy drinks frequently used as mixers for alcoholic beverages. However, the combined effects of TAU and ethanol (EtOH) on behavioral parameters such as aggression are poorly understood. Considering that zebrafish is a suitable vertebrate to assess agonistic behaviors using noninvasive protocols, we investigate whether TAU modulates EtOH-induced aggression in zebrafish using the mirror-induced aggression (MIA) test. Since body color can be altered by pharmacological agents and may be indicative of emotional state, we also evaluated the actions of EtOH and TAU on pigment response. Fish were acutely exposed to TAU (42, 150, and 400mg/L), EtOH (0.25%), or cotreated with both molecules for 1h and then placed in the test apparatus for 6min. EtOH, TAU 42, TAU 400, TAU 42/EtOH and TAU 400/EtOH showed increased aggression, while 150mg/L TAU only increased the latency to attack the mirror. This same concentration also prevented EtOH-induced aggression, suggesting that it antagonizes the effects of acute alcohol exposure. Representative ethograms revealed the existence of different aggressive patterns and our results were confirmed by an index used to estimate aggression in the MIA test. TAU did not alter pigment intensity, while EtOH and all cotreated groups presented a substantial increase in body color. Overall, these data show a biphasic effect of TAU on EtOH-induced aggression of zebrafish, which is not necessarily associated with changes in body color.

The role of taurine on anxiety-like behaviors in zebrafish: A comparative study using the novel tank and the light-dark tasks.

Taurine (TAU) is an amino sulfonic acid with several functions in central nervous system. Mounting evidence suggests that it acts in osmoregulation, neuromodulation and also as an inhibitory neurotransmitter. However, the effects of TAU on behavioral functions, especially on anxiety-related parameters, are limited. The adult zebrafish is a suitable model organism to examine anxiety-like behaviors since it presents neurotransmitter systems and behavioral functions evolutionary conserved. Anxiety in zebrafish can be measured by different tasks, analyzing the habituation to novelty, as well as the response to brightly lit environments. The aim of this study was to investigate whether acute TAU treatment alters anxiety-like behavior in zebrafish using the novel tank and the light-dark tests. Fish were individually treated with TAU (42, 150, and 400mg/L) for 1h and the behaviors were further analyzed for 6min in the novel tank or in the light-dark test. Control fish were handled in a similar manner, but kept only in home tank water. Although TAU did not alter locomotor and vertical activities, all concentrations significantly increased shuttling and time spent in lit compartment. Moreover, TAU 150 group showed a significant decrease in the number of risk assessment episodes. Overall, these data suggest that TAU exerts an anxiolytic-like effect in zebrafish and the comparative analysis of behavior using different tasks is an interesting strategy for neuropsychiatric studies related to anxiety in this species.

Conspecific alarm substance differently alters group behavior of zebrafish populations: Putative involvement of cholinergic and purinergic signaling in anxiety- and fear-like responses

Graphical abstract Figure. No Caption available. HighlightsAlarm substance induces fear in zebrafish shoals of two populations.Leo shoals show pronounced basal defensive responses in comparison to WT fish.Only WT shows enhanced shoaling and diving response triggered by alarm substance.The cholinergic and purinergic systems may play a role in defensive behaviors. ABSTRACT The zebrafish (Danio rerio) is an emergent model organism for assessing fear and anxiety‐like phenotypes. The short fin wild type (WT), and leopard (leo) are two zebrafish populations that present several behavioral differences, in which leo displays pronounced defensive responses. Mounting evidence suggests a modulatory role for cholinergic and purinergic signaling in fear and anxiety, but the involvement of these neurotransmitter systems in the behavioral profile of zebrafish is obscure. Here we tested whether the acute exposure to conspecific alarm substance (AS), an experimental protocol that induces fear, alters shoaling behavior, diving response, acetylcholinesterase (AChE) activity, and nucleotide hydrolysis in brain tissue of WT and leo. When four fish were concomitantly exposed to AS extracted from a donor fish of similar phenotype, both populations presented a significant increase of erratic movements without changes in freezing bouts. An increased shoal cohesion and a decreased vertical distribution were observed only in WT exposed to AS. The respective population also revealed a significant increase in AChE and ecto‐5′‐nucleotidase activities after the exposure period. The comparison of basal endpoints between populations showed that leo displays a higher social cohesion, few vertical transitions and enhanced AChE and ecto‐5′‐nucleotidase activities. In conclusion, we suggest that the effects of AS on defensive behaviors depend on the population, indicating the existence of distinct neurochemical mechanisms involved. Furthermore, this report shows the first evidence of a potential role of cholinergic and purinergic systems in fear‐ and anxiety‐like responses of zebrafish populations.

Different effects of caffeine on behavioral neurophenotypes of two zebrafish populations

ABSTRACT Caffeine is a substance present in several foods and drinks of common western diet. Although high caffeine concentrations induce anxiogenic properties in various species, the influence of the different baselines of anxiety levels on caffeine‐mediated responses is poorly understood. The short‐fin wild‐type (WT) and leopard (leo) zebrafish populations present significant behavioral differences, in which leo shows exacerbated anxiety‐like responses. Since behavioral neurophenotyping may be easily assessed in adult zebrafish by associating temporal and spatial three‐dimensional reconstructions of locomotion, we investigated the effects of caffeine on exploration and anxiety‐like behavior of WT and leo zebrafish. Moreover, the whole‐body cortisol content was assessed in the absence and presence of caffeine. For this purpose, animals were acutely exposed to caffeine (25, 50, 100 and 200 mg/L) for 15 min and further tested in the novel tank. Endpoint data and 3D reconstruction plots revealed that caffeine was anxiogenic in both WT and leo populations by altering vertical swimming, freezing, and erratic movements depending on the concentration. Prominent anxiogenic effects during habituation to novelty were observed in WT, suggesting a fundamental role of the phenotype in caffeine‐mediated neurobehavioral responses. Although untreated leo showed higher baseline cortisol levels than control WT, caffeine increased whole‐body cortisol in both populations. Moreover, caffeine induced aberrant swimming profiles in WT and leo following 200 mg/L exposure, which could reflect nonspecific toxicity and/or seizure‐like behaviors. Collectively, our novel findings show that caffeine effects in zebrafish differ in a population‐dependent manner. HIGHLIGHTSBehavioral effects of caffeine were tested in WT and leo zebrafish populations.Behavioral neurophenotyping was analyzed by endpoints and 3D swimming traces.Caffeine increased anxiogenic‐like behaviors and whole‐body cortisol of zebrafish.Caffeine prominently impaired the habituation to novelty in WT.High caffeine concentrations induced abnormal behavior in zebrafish.

Azadirachtin, a neem‐derived biopesticide, impairs behavioral and hematological parameters in carp (Cyprinus carpio)

Azadirachtin (Aza) is a promisor biopesticide used in organic production and aquaculture. Although this compound is apparently safe, there is evidence that it may have deleterious effects on fish. Behavioral and hematological tests are grouped into a set of parameters that may predict potential toxicity of chemical compounds. Here, we investigate the effects of Aza, in the commercial formulation Neenmax™, on carp (Cyprinus carpio) by defining LC50 (96 h), and testing behavioral and hematological parameters. In our study, LC50 was estimated at 80 μL/L. We exposed carp to Aza at 20, 40, and 60 μL/L, values based on 25, 50, and 75% of LC50, respectively. At 60 μL/L, Aza promoted significant changes in several parameters, increasing the distance traveled and absolute turn angle. In addition, the same concentration decreased the time spent immobile and the number of immobile episodes. Hematological parameters, such as hematocrit, hemoglobin, hematimetrics index, and red cell distribution, were decreased at 60 μL/L Aza exposure. In conclusion, our study demonstrates that 60 μL/L Aza altered locomotor activity, motor pattern, and hematological parameters, suggesting potential toxicity to carp after acute exposure. In addition, this is the first report that evaluates the actions of a chemical contaminant using automated behavioral tracking of carp, which may be a useful tool for assessing the potential toxicity of biopesticides in conjunction with hematological tests.

Modulatory role of conspecific alarm substance on aggression and brain monoamine oxidase activity in two zebrafish populations.

&NA; Aversive conditions can elicit fear and the subsequent activation of the sympathetic nervous system induces ‘fight or flight’ responses. Previous unpleasant experiences may trigger fear‐induced aggression and heightened aggression is a behavioral phenotype associated to various psychopathologies. Since the conspecific alarm substance (CAS) acts as chemical cue that elicits fear in fish species, we evaluated whether acute and chronic CAS exposures modulate aggression in wild‐type (WT) and leopard (leo) zebrafish using the mirror‐induced aggression (MIA) test. Because monoamines influence mood and behavior, we also assessed the effects of CAS on brain Z‐MAO activity. CAS was isolated from phenotypically similar donor fish and 3.5 mL/L was used for the experiments. In the acute protocol, fish were tested following a single CAS exposure (5 min). The chronic exposure consisted of exposing the animals once daily (5 min) for 7 consecutive days, with a subsequent test on the 8th day. CAS acutely increased aggression and decreased Z‐MAO activity in both populations. Conversely, chronic CAS exposure reduced aggression and inhibited locomotion without affecting Z‐MAO. Differently than WT, leo showed decreased absolute turn angle and increased latency to attack the mirror following the chronic exposure. At baseline conditions, WT were more active, aggressive, and had a lower brain Z‐MAO activity than leo. Overall, we suggest a distinct acute and chronic effect of CAS on aggression and a possible involvement of brain Z‐MAO in aggressive behaviors. Moreover, the use of different zebrafish populations could serve as emergent tools to investigate the neurobehavioral bases of fear‐induced aggression. Graphical abstract Representative data following acute and repeated CAS exposure on aggressive behavior and brain Z‐MAO activity of WT and leo zebrafish populations. The ethograms show distance traveled, aggressive episodes, and duration of aggressive behaviors measured in the MIA test. Figure. No caption available. HighlightsCAS modulates aggression and brain Z‐MAO of WT and leo zebrafish populations.Baseline conditions reflect increased aggression and lower Z‐MAO activity in WT.Acute CAS exposure exacerbates aggression and decreases Z‐MAO activity of zebrafish.Chronic CAS exposure impairs locomotion without changing Z‐MAO activity.Motor patterns in leo are altered following repeated CAS exposure.

Ketamine modulates aggressive behavior in adult zebrafish

Ketamine is a non-competitive glutamatergic antagonist that induces analgesia and anesthesia. Although ketamine displays anxiolytic and antidepressant properties, it may induce pro-psychosis and hallucinogen effects, as well as stereotypic behaviors following acute administration at sub-anesthetic doses. Since heightened aggression is maladaptive and may comorbid with various neuropsychiatric disorders, we aimed to investigate whether ketamine modulates aggressive behavior in adult zebrafish. Fish were acutely exposed to 2, 20, and 40 mg/L ketamine for 20 min and their locomotion, exploratory activity, and aggression towards mirror were further assessed. Ketamine (2 mg/L) increased aggression-related phenotypes, while 20 and 40 mg/L reduced aggression and elicited stereotypic behaviors by causing hyperlocomotion, altering motor patterns, and increasing circling behavior at the higher concentration tested. Collectively, our data expand the utility of zebrafish models to investigate the influence of sub-anesthetic concentrations of ketamine on aggression behavior domain in translational neuropsychiatric research field.

Single pentylenetetrazole exposure increases aggression in adult zebrafish at different time intervals

Epilepsy is characterized by abnormal and recurrent hyperexcitability in brain cells. Various comorbidities are associated with epilepsy, including irritability and aggressive behavior. Aggression is a negative effect observed in epileptic patients that may be harmful to other individuals, impairing social relations. Thus, developing novel experimental models to assess behavioral phenotypes that may comorbid with neurological disorders are of great interest. Here, we investigate whether pentylenetetrazole (PTZ) increases aggression in zebrafish following a single exposure. Animals were exposed to 10 mM PTZ for 20 min and aggression-towards mirror was measured at different time intervals after recovering period (1 h, 3 h, 6 h, 24 h, 48 h, and 72 h). We observed that zebrafish showed exacerbated aggression, as well as an increased number of entries in the virtual conspecific area from 1 h to 48 h after PTZ. However, no behavioral differences were observed after 72 h. Overall, our novel findings show that a single PTZ exposure evokes aggression in a time-dependent manner, reinforcing the use of zebrafish models to explore epilepsy-related comorbidities.

Taurine modulates acute ethanol-induced social behavioral deficits and fear responses in adult zebrafish

Ethanol (EtOH) is a central nervous system (CNS) depressant drug that modifies various behavioral domains (i.e., sociability, aggressiveness, and memory) by promoting disinhibition of punished operant behavior and neurochemical changes. Taurine (TAU) is a β-amino sulfonic acid with pleiotropic roles in the brain. Although exogenous TAU is found in energy drinks and often mixed with alcohol in beverages, the putative risks of mixing TAU and EtOH are poorly explored. Here, we investigated whether TAU modulates social and fear responses by assessing shoaling behavior, preference for conspecifics, and antipredatory behavior of adult zebrafish acutely exposed to EtOH. Zebrafish shoals (4 fish per shoal) were exposed to water (control), TAU (42, 150, and 400 mg/L), 0.25% (v/v) EtOH alone or in association with TAU for 1 h, and their behaviors were analyzed at different time intervals (0-5 min, 30-35 min, and 55-60 min). The effects of TAU and EtOH were further tested in a social preference test and during exposure to a predator. Both EtOH and TAU co-treated fish showed a higher shoal dispersion, while TAU 400/EtOH group shoal area had a similar profile when compared to control. However, in the social preference test, TAU 400/EtOH impaired the seeking for conspecifics. Regarding fear-like behaviors, TAU-cotreated fish showed a prominent reduction in risk assessments when compared to EtOH alone. Overall, we demonstrate that TAU modulates EtOH-induced changes in different behavioral domains, suggesting a complex relationship between social and fear-like responses.