Anxiety-like Behaviour In Zebrafish Research Articles

The influence of exercise on anxiety-like behavior in zebrafish (Danio rerio)

In non-human mammals, exercise has been shown to decrease anxiety-like behavior. Conversely, a number of studies have reported no effect or even an increase in anxiety-like behavior after exercise, however, inconsistent training regimes and behavioral paradigms across studies may be confounding the results. Zebrafish (Danio rerio) are a well-established animal model in neurobehavioral research, and have the potential to shed new insight into the effects of exercise on anxiety-like behavior where previous research has been limited, due to the ability to precisely control intensity and duration of exercise, and the validation of tests for measuring different aspects of anxiety-like behaviors. In the current study, fish were split between two treatment groups; Exercised and Control. Fish in the exercised condition were aerobically challenged (max water velocity: 0.5 m/s) using a swim tunnel one hour a day, five days a week, for six weeks. Control fish spent an equal amount of time in the swim tunnel but were not aerobically challenged (max water velocity: 0.05 m/s). After six weeks, all fish were tested individually in two standard complimentary anxiety tests for zebrafish: the novel tank test and the light-dark test. Exercised fish exhibited reduced anxiety-like behaviors in the novel tank test; they spent more time in the top and were quicker to enter the top of a novel tank compared to Control fish. In addition, Exercised fish spent more time in the light compartment of the light-dark test compared to Control fish. Our results demonstrate the beneficial effect of exercise on anxiety-like behavior in zebrafish.

Effects of alprazolam treatment on anxiety-like behavior induced by color stimulation in adult zebrafish

It has been reported that the use of certain stimuli can lead to anxiety-like behavior in zebrafish. Moreover, visual stimulation of zebrafish is becoming a popular tool. Here we evaluated the effects of six colors combinations and alprazolam, a benzodiazepine which is widely used in the treatment of anxiety disorders, on the behavior of adult zebrafish in a two-chambered apparatus, which chambers were illuminated by red/yellow, green/blue, red/green, green/yellow, red/blue and blue/yellow light. The following parameters were measured: time spent in the zone, number of entries to the zone, time of freezing, distance traveled and average speed in the zone. We report that the adult zebrafish spent more time in the red zone compared to yellow or green as well as in the yellow or blue compared to green. The zebrafish displayed a concomitant increase in time freezing in the red zone compared to yellow or green as well as in the yellow or blue compared to green. Moreover, average speed was decreased in the red zone compared to yellow or green and in the yellow zone compared to green. Treatment with alprazolam significantly affected the behavior of the zebrafish, e.g., following alprazolam administration time spent in the zone and time freezing were longer in the green zone than in red. Based on these observations, we suggest that light color combinations could be effective to manipulate zebrafish behavior and could be useful in neuropsychopharmacological studies, perhaps to study anxiety-like behavior and the effects of anxiolytic agents.

Behavioral Changes Over Time Following Ayahuasca Exposure in Zebrafish.

The combined infusion of Banisteriopsis caapi stem and Psychotria viridis leaves, known as ayahuasca, has been used for centuries by indigenous tribes. The infusion is rich in N, N-dimethyltryptamine (DMT) and monoamine oxidase inhibitors, with properties similar to those of serotonin. Despite substantial progress in the development of new drugs to treat anxiety and depression, current treatments have several limitations. Alternative drugs, such as ayahuasca, may shed light on these disorders. Here, we present time-course behavioral changes induced by ayahuasca in zebrafish, as first step toward establishing an ideal concentration for pre-clinical evaluations. We exposed adult zebrafish to five concentrations of the ayahuasca infusion: 0 (control), 0.1, 0.5, 1, and 3 ml/L (n = 14 each group), and behavior was recorded for 60 min. We evaluated swimming speed, distance traveled, freezing and bottom dwelling every min for 60 min. Swimming speed and distance traveled decreased with an increase in ayahuasca concentration while freezing increased with 1 and 3 ml/L. Bottom dwelling increased with 1 and 3 ml/L, but declined with 0.1 ml/L. Our data suggest that small amounts of ayahuasca do not affect locomotion and reduce anxiety-like behavior in zebrafish, while increased doses of the drug lead to crescent anxiogenic effects. We conclude that the temporal analysis of zebrafish behavior is a sensitive method for the study of ayahuasca-induced functional changes in the vertebrate brain.

Modafinil decreases anxiety-like behaviour in zebrafish.

Modafinil (2-((diphenylmethyl)sulfinyl)acetamide), a selective dopamine and norepinephrine transporter inhibitor, is most commonly prescribed for narcolepsy but has gained recent interest for treating a variety of disorders. Zebrafish (Danio rerio) are becoming a model of choice for pharmacological and behavioural research. To investigate the behavioural effects of modafinil on anxiety, we administered doses of 0, 2, 20, and 200 mg/L for 30 minutes then tested zebrafish in the novel approach test. In this test, the fish was placed into a circular arena with a novel object in the center and motion-tracking software was used to quantify the time the fish spent in the outer area of the arena (thigmotaxis zone), middle third of the arena (transition zone) and center of the arena, as well as total distance traveled, immobility and meandering. Modafinil caused a decrease in time spent in the thigmotaxis zone and increased time spent in the transition zone across all doses. Modafinil did not significantly alter the time spent in the center zone (near the novel object), the distance moved, meandering, or the duration of time spent immobile. We also validated this test as a measure of anxiety with the administration of ethanol (1%) which decreased time spent in the thigmotaxis zone and increased time spent in the transition zone. These results suggest that modafinil decreases anxiety-like behaviour in zebrafish.

Water column depth and light intensity modulate the zebrafish preference response in the black/white test

Currently, the black/white preference test has been used to evaluate anxiety-like behaviors in zebrafish. However, several inconsistent results have been reported across literature. Since animal behavior can be influenced by several environmental factors, the main goal of the present study was to investigate the influence of different water column depths and light intensities on zebrafish behavioral responses in the black/white test. On a 4cm water column depth, animals spent more time in the black than in the white compartment. However, when animals were tested in an 8cm water column, no significant difference was found. Using an inclined acrylic floor inside the aquarium, animals spent more time in the deep compartment when this was black. However, there is no difference in time spent in each compartment when the deeper compartment was white. For light intensity test, animals showed preference for the white compartment only when both compartments were illuminated with 100lx. For the others illumination settings, there was no difference in the compartment preference. In conclusion, our results suggest that variations in water column depth and light intensity can modulate zebrafish preference in the black/white test. These variations may be implicated in the discrepancies observed in literature.

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.

L-Scoulerine attenuates behavioural changes induced by methamphetamine in zebrafish and mice

Methamphetamine (METH), a substance with a high potential for abuse and addiction, is a serious worldwide public health problem. METH addicts often show extreme paranoia, anxiety, and depression. Thus, there is no effective medication for the treatment of METH-induced abnormalities. In the present study, we investigated the effects of l-Scoulerine (l-SLR), a tetrahydroprotoberberine (THPBS) alkaloid, on METH-induced anxiety-like behaviour in zebrafish and METH-induced addictive behavior in mice. In the novel tank test, acute administration of METH (2mg/L) induced a significant decrease in the number of total vertical transitions and time spent in the upper zone. Moreover, METH produced significant avoidance behaviour showing increased swimming time in the central area and high speed movement in the mirror area in the mirror stimulation test; these anxiety-like changes were attenuated by l-SLR. Chronic administration of METH (2mg/kg) produced a steady increase in locomotor activity and conditioned place preference in mice. l–SLR (5mg/kg) failed to reduce acute METH-induced hyperlocomotion, but attenuated chronic METH-induced behavioural sensitization and significantly blocked the expression of conditioned place preference induced by METH in mice. The present study suggests that l-SLR may be a promising agent for the treatment of addiction and anxiety induced by METH.

Putative involvement of the nitrergic system on the consolidation, but not initiation, of behavioral sensitization after conspecific alarm substance in zebrafish

Stressful manipulations can sensitize the behavior of an organism, increasing anxiety-like behavior after a delay; this long-term stress sensitization can represent the pathophysiological basis of trauma- and stress-related disorders (TRSDs), of which the most prevalent is post-traumatic stress disorder (PTSD). A role for the glutamate–nitric oxide pathway in this sensitization is implied by behavioral, neurophysiological and genomic data on different species. Here, we report on the long-term sensitization of anxiety-like behavior in zebrafish and the possible participation of nitric oxide in this process. Zebrafish exposed to a conspecific alarm substance (AS) show increased anxiety-like behavior at least 24h after stimulus delivery. Blocking nitric oxide synthesis with l-NAME (5mg/kg) 30min, but not 90min, after AS exposure blocks the sensitization of scototaxis and risk assessment, while treatment 90min after exposure blocks the sensitization of thigmotaxis and erratic swimming; l-NAME was not effective when administered 30min before AS exposure. These data suggest a participation of nitric oxide in the consolidation, but not in the initiation, of behavioral sensitization after predator threat.

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.

Involvement of the α1-adrenoceptor in sleep–waking and sleep loss-induced anxiety behavior in zebrafish

Sleep is a universal phenomenon in vertebrates, and its loss affects various behaviors. Independent studies have reported that sleep loss increases anxiety; however, the detailed mechanism is unknown. Because sleep deprivation increases noradrenalin (NA), which modulates many behaviors and induces patho-physiological changes, this study utilized zebrafish as a model to investigate whether sleep loss-induced increased anxiety is modulated by NA. Continuous behavioral quiescence for at least 6s was considered to represent sleep in zebrafish; although some authors termed it as a sleep-like state, in this study we have termed it as sleep. The activity of fish that signified sleep–waking was recorded in light–dark, during continuous dark and light; the latter induced sleep loss in fish. The latency, number of entries, time spent and distance travelled in the light chamber were assessed in a light–dark box test to estimate the anxiety behavior of normal, sleep-deprived and prazosin (PRZ)-treated fish. Zebrafish showed increased waking during light and complete loss of sleep upon continuous exposure to light for 24h. PRZ significantly increased sleep in normal fish. Sleep-deprived fish showed an increased preference for dark (expression of increased anxiety), and this effect was prevented by PRZ, which increased sleep as well. Our findings suggest that sleep loss-induced anxiety-like behavior in zebrafish is likely to be mediated by NA’s action on the α1-adrenoceptor.

Caffeine neuroprotects against dexamethasone-induced anxiety-like behaviour in the Zebrafish (Danio rerio)

The early-life stress has critical impact on brain development which can lead to long-term effects on brain functions during adulthood. It has been reported that caffeine possesses a protective effect in neurodegenerative diseases. Thus, this study investigates the potential of caffeine to protect brain functions from adverse effects due to stress exposure during early-life development in the male zebrafish. In the first part of this study, synthetic glucocorticoid, dexamethasone (DEX) (2–200mg/L for 24h) was used to induce stress effects in the zebrafish larvae from 4 to 5days post-fertilisation (dpf) and the effect of DEX administration on zebrafish larvae on anxiety-like behaviour during adulthood in novel tank test was investigated. Next, the possible protective effect of caffeine pre-treatment (5–50mg/L for 24h from 3 to 4dpf) before DEX administration was studied. DEX-treated adult male zebrafish showed higher anxiety levels in behavioural tests, as seen in longer latency to enter the top part of the tank, lower transition numbers between the top and bottom parts with more time spent at the bottom and lesser time spent at the top and lower distance travelled at top part. The effect of DEX on anxiety-like behaviour was dose-dependent. Importantly, adult male zebrafish pre-treated with caffeine before DEX treatment did not show any anxiety-like behaviour. These results show that exposure to stress during early-life leads to anxiety-like behaviour in the adult male zebrafish but pre-treatment with caffeine protects from stress-induced anxiety.

Differences of acute versus chronic ethanol exposure on anxiety-like behavioral responses in zebrafish

Zebrafish, a vertebrate model organism amenable to high throughput screening, is an attractive system to model and study the mechanisms underlying human diseases. Alcoholism and alcoholic medical disorders are among the most debilitating diseases, yet the mechanisms by which ethanol inflicts the disease states are not well understood. In recent years zebrafish behavior assays have been used to study learning and memory, fear and anxiety, and social behavior. It is important to characterize the effects of ethanol on zebrafish behavioral repertoires in order to successfully harvest the strength of zebrafish for alcohol research. One prominent effect of alcohol in humans is its effect on anxiety, with acute intermediate doses relieving anxiety and withdrawal from chronic exposure increasing anxiety, both of which have significant contributions to alcohol dependence. In this study, we assess the effects of both acute and chronic ethanol exposure on anxiety-like behaviors in zebrafish, using two behavioral paradigms, the Novel Tank Diving Test and the Light/Dark Choice Assay. Acute ethanol exposure exerted significant dose-dependent anxiolytic effects. However, withdrawal from repeated intermittent ethanol exposure disabled recovery from heightened anxiety. These results demonstrate that zebrafish exhibit different anxiety-like behavioral responses to acute and chronic ethanol exposure, which are remarkably similar to these effects of alcohol in humans. Because of the accessibility of zebrafish to high throughput screening, our results suggest that genes and small molecules identified in zebrafish will be of relevance to understand how acute versus chronic alcohol exposure have opposing effects on the state of anxiety in humans.