Seizures Induced by Pentylenetetrazole in the Adult Zebrafish: A Detailed Behavioral Characterization

Pentylenetetrazole (PTZ) is a common convulsant agent used in animal models to investigate the mechanisms of seizures. Although adult zebrafish have been recently used to study epileptic seizures, a thorough characterization of the PTZ-induced seizures in this animal model is missing. The goal of this study was to perform a detailed temporal behavior profile characterization of PTZ-induced seizure in adult zebrafish. The behavioral profile during 20 min of PTZ immersion (5, 7.5, 10, and 15 mM) was characterized by stages defined as scores: (0) short swim, (1) increased swimming activity and high frequency of opercular movement, (2) erratic movements, (3) circular movements, (4) clonic seizure-like behavior, (5) fall to the bottom of the tank and tonic seizure-like behavior, (6) death. Animals exposed to distinct PTZ concentrations presented different seizure profiles, intensities and latencies to reach all scores. Only animals immersed into 15 mM PTZ showed an increased time to return to the normal behavior (score 0), after exposure. Total mortality rate at 10 and 15 mM were 33% and 50%, respectively. Considering all behavioral parameters, 5, 7.5, 10, and 15 mM PTZ, induced seizures with low, intermediate, and high severity, respectively. Pretreatment with diazepam (DZP) significantly attenuated seizure severity. Finally, the brain PTZ levels in adult zebrafish immersed into the chemoconvulsant solution at 5 and 10 mM were comparable to those described for the rodent model, with a peak after a 20-min of exposure. The PTZ brain levels observed after 2.5-min PTZ exposure and after 60-min removal from exposure were similar. Altogether, our results showed a detailed temporal behavioral characterization of a PTZ epileptic seizure model in adult zebrafish. These behavioral analyses and the simple method for PTZ quantification could be considered as important tools for future investigations and translational research.     

Light/dark transition test for mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing. Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.     

Homebase behavior of zebrafish in novelty-based paradigms

Zebrafish (Danio rerio) are emerging as a promising model species in neuroscience research. Many traditional rodent behavioral paradigms may be adapted for zebrafish testing. Exposing zebrafish to three different “open field” tanks for 30 min, we showed that fish display robust homebase behavior, in which one area of the tank is chosen as a preferred point of reference during the test, which the fish frequently return to and spend a longer duration in. This phenotype strikingly resembles rodent homebase behavior, confirming that both species use homebases as “reference points” for their exploration. Our study introduces a simple method for zebrafish homebase phenotyping, and further supports the utility of these fish in neurobehavioral and cognitive research.     

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 .     

Mitragynine attenuates withdrawal syndrome in morphine-withdrawn zebrafish

A major obstacle in treating drug addiction is the severity of opiate withdrawal syndrome, which can lead to unwanted relapse. Mitragynine is the major alkaloid compound found in leaves of Mitragyna speciosa, a plant widely used by opiate addicts to mitigate the harshness of drug withdrawal. A series of experiments was conducted to investigate the effect of mitragynine on anxiety behavior, cortisol level and expression of stress pathway related genes in zebrafish undergoing morphine withdrawal phase. Adult zebrafish were subjected to two weeks chronic morphine exposure at 1.5 mg/L, followed by withdrawal for 24 hours prior to tests. Using the novel tank diving tests, we first showed that morphine-withdrawn zebrafish display anxiety-related swimming behaviors such as decreased exploratory behavior and increased erratic movement. Morphine withdrawal also elevated whole-body cortisol levels, which confirms the phenotypic stress-like behaviors. Exposing morphine-withdrawn fish to mitragynine however attenuates majority of the stress-related swimming behaviors and concomitantly lower whole-body cortisol level. Using real-time PCR gene expression analysis, we also showed that mitragynine reduces the mRNA expression of corticotropin releasing factor receptors and prodynorphin in zebrafish brain during morphine withdrawal phase, revealing for the first time a possible link between mitragynine's ability to attenuate anxiety during opiate withdrawal with the stress-related corticotropin pathway.     

Sustained Effects of Developmental Exposure to Ethanol on Zebrafish Anxiety-Like Behaviour

In zebrafish developmentally exposed to ambient ethanol (20mM-50mM) 1–9 days post fertilization (dpf), the cortisol response to stress has been shown to be significantly attenuated in larvae, juveniles and 6 month old adults. These data are somewhat at variance with similar studies in mammals, which often show heightened stress responses. To test whether these cortisol data correlate with behavioural changes in treated animals, anxiety-like behaviour of zebrafish larvae (9dpf and 10dpf) and juveniles (23dpf) was tested in locomotor assays designed to this end. In open field tests treated animals were more exploratory, spending significantly less time at the periphery of the arena. Behavioural effects of developmental exposure to ethanol were sustained in 6-month-old adults, as judged by assessment of thigmotaxis, novel tank diving and scototaxis. Like larvae and juveniles, developmentally treated adults were generally more exploratory, and spent less time at the periphery of the arena in thigmotaxis tests, less time at the bottom of the tank in the novel tank diving tests, and less time in the dark area in scototaxis tests. The conclusion that ethanol-exposed animals showed less anxiety-like behaviour was validated by comparison with the effects of diazepam treatment, which in thigmotaxis and novel tank diving tests had similar effects to ethanol pretreatment. There is thus a possible link between the hypophyseal-pituitary-interrenal axis and the behavioural actions of developmental ethanol exposure. The mechanisms require further elucidation.     

Chronic Treatment with Paraquat Induces Brain Injury,Changes in Antioxidant Defenses System,and Modulates Behavioral Functions in Zebrafish

Paraquat (PQ) administration consists in a chemical model that mimics phenotypes observed in Parkinson’s disease (PD), due to its ability to induce changes in dopaminergic system and oxidative stress. The aim of this study was to evaluate the actions of PQ in behavioral functions of adult zebrafish and its influence on oxidative stress biomarkers in brain samples. PQ (20 mg/kg) was administered intraperitoneally with six injections for 16 days (one injection every 3 days). PQ-treated group showed a significant decrease in the time spent in the bottom section and a shorter latency to enter the top area in the novel tank test. Moreover, PQ-exposed fish showed a significant decrease in the number and duration of risk assessment episodes in the light–dark test, as well as an increase in the agonistic behavior in the mirror-induced aggression (MIA) test. PQ induced brain damage by decreasing mitochondrial viability. Concerning the antioxidant defense system, PQ increased catalase (CAT) and glutathione peroxidase (GPx) activities, as well as the non-protein sulfhydryl content (NPSH), but did not change ROS formation and decreased lipid peroxidation. We demonstrate, for the first time, that PQ induces an increase in aggressive behavior, alters non-motor patterns associated to defensive behaviors, and changes redox parameters in zebrafish brain. Overall, our findings may serve as useful tools to investigate the interaction between behavioral and neurochemical impairments triggered by PQ administration in zebrafish.     

Measuring behavioral and endocrine responses to novelty stress in adult zebrafish

Several behavioral assays are currently used for high-throughput neurophenotyping and screening of genetic mutations and psychotropic drugs in zebrafish (Danio rerio). In this protocol, we describe a battery of two assays to characterize anxiety-related behavioral and endocrine phenotypes in adult zebrafish. Here, we detail how to use the 'novel tank' test to assess behavioral indices of anxiety (including reduced exploration, increased freezing behavior and erratic movement), which are quantifiable using manual registration and computer-aided video-tracking analyses. In addition, we describe how to analyze whole-body zebrafish cortisol concentrations that correspond to their behavior in the novel tank test. This protocol is an easy, inexpensive and effective alternative to other methods of measuring stress responses in zebrafish, thus enabling the rapid acquisition and analysis of large amounts of data. As will be shown here, fish anxiety-like behavior can be either attenuated or exaggerated depending on stress or drug exposure, with cortisol levels generally expected to parallel anxiety behaviors. This protocol can be completed over the course of 2 d, with a variable testing duration depending on the number of fish used.     

Housing mice in the individually ventilated or open cages—Does it matter for behavioral phenotype?

Individually ventilated caging (IVC) systems for rodents are increasingly common in laboratory animal facilities. However, the impact of such substantial change in housing conditions on animal physiology and behavior is still debated. Most importantly, there arise the questions regarding reproducibility and comparison of previous or new phenotypes between the IVC and open cages. The present study was set up for detailed and systematic comparison of behavioral phenotypes in male and female mice of three widely used inbred strains (C57BL/6JRccHsd, DBA/2JRccHsd, 129S2/SvHSd) after being kept in two housing environments (IVC and open cages) for 6 weeks (since 4 weeks of age) before behavioral testing. The tests addressed exploratory, anxiety‐like and stress‐related behavior (light‐dark box, open field, forced swim test, stress‐induced hyperthermia), social approach and species‐specific behavior (nest building, marble burying). In all tests, large and expected strain differences were found. Somewhat surprisingly, the most striking effect of environment was found for basal body temperature and weight loss after one night of single housing in respective cages. In addition, the performance in light‐dark box and open field was affected by environment. Several parameters in different tests showed significant interaction between housing and genetic background. In summary, the IVC housing did not invalidate the well‐known differences between the mouse strains which have been established by previous studies. However, within the strains the results can be influenced by sex and housing system depending on the behavioral tasks applied. The bottom‐line is that the environmental conditions should be described explicitly in all publications.     

Repeated measures of shoaling tendency in zebrafish (Danio rerio) and other small teleost fishes

This protocol details a method for constructing and using both a holding tank and a test tank to assess the shoaling tendency of zebrafish and other teleosts. The test tank consists of a central compartment with two side compartments, one of which contains a shoal of stimulus fish. The focal fish is released and the amount of time spent associating with the stimulus shoal is recorded over a 10-min period. A holding tank enables individual fish to be accurately retested with a minimum of stress. Testing takes around 15 min per fish.     

Behavioral states in space and time: understanding landscape use by an invasive mammal

Animal movement models can be used to understand species behavior and assist with implementation of management activities. We explored behavioral states of an invasive wild pig (Sus scrofa) population that recently colonized central Michigan, USA, 2014–2018. To quantify environmental factors related to wild pig movement ecology and spatio-temporal landscape use, we predicted wild pig behavioral states relative to land cover type, landscape structure (i.e., edge and patch cohesion), and weather conditions. We used global positioning system (GPS)-collars and monitored 8 wild pigs from 2014–2018. We fit local convex hulls and calculated movement metrics revealing 3 wild pig behavioral states (resting, exploratory, and relocating) and constructed a 3-level model to predict behavioral state probabilities relative to biotic and abiotic conditions. Probabilities of exploratory and resting behaviors were higher nearer to riparian and open herbaceous cover types (oftentimes emergent marsh), indicating that these cover types provided security cover during activity and bedding. Hard mast cover types had a strong positive association with relocating behaviors. More cohesive patches of agriculture and shrub cover types were associated with higher probabilities of exploratory behaviors, while resting was more likely in continuous patches of agriculture (mostly mid-summer corn). The probability of exploratory behaviors increased exponentially with warming ambient temperature. Our results may be used by managers to develop control strategies conducive to landscape and environmental conditions where the likelihood of encountering wild pigs is highest or targeting wild pigs when in a behavioral state most vulnerable to a particular removal technique.     

Movement, technology and discovery in the zebrafish

Zebrafish provide unique opportunities for optogenetic studies of behavior. Here, we review the most recent work using optogenetic and imaging approaches to study the neuronal circuits controlling movements in the transparent zebrafish. Specifically, we focus on what we have learned from zebrafish about neuronal migration, network formation and behavioral control, and what the future may hold.     

The function of wall-following behaviors in the Mexican blind cavefish and a sighted relative,the Mexican tetra (<Emphasis Type="Italic">Astyanax</Emphasis>)

Mexican blind cavefish exhibit an unconditioned wall-following behavior in response to novel environments. Similar behaviors have been observed in a wide variety of animals, but the biological significance and evolutionary history of this behavior are largely unknown. In this study, the behaviors of Mexican blind cavefish (Astyanax sp.) and sighted Mexican tetra (Astyanax mexicanus) were videotaped after fish were introduced into a novel environment under dark (infrared) or well-lit conditions. Under dark conditions, both sighted and blind morphs exhibited wall-following behaviors with subtle but significant differences. Blind morphs swam more nearly parallel to the wall, exhibited greater wall-following continuity and reached higher levels of sustained swimming speeds more quickly than sighted morphs. In contrast, sighted morphs in the light remained motionless near the wall for long periods of time or moved slowly around the center of the tank without entraining to the walls. These results are consistent with the idea that wall-following is a shared, primitive trait that serves an exploratory function under dark conditions to compensate for the absence of vision. This behavior has become more honed in blind morphs for exploratory purposes—in large part due to the enhanced, active-flow sensing abilities of the lateral line.     

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing. Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.     

Measures of anxiety in zebrafish (Danio rerio): dissociation of black/white preference and novel tank test

The effects of wall color stimuli on diving, and the effects of depth stimuli on scototaxis, were assessed in zebrafish. Three groups of fish were confined to a black, a white, or a transparent tank, and tested for depth preference. Two groups of fish were confined to a deep or a shallow tank, and tested for black-white preference. As predicted, fish preferred the deep half of a split-tank over the shallow half, and preferred the black half of a black/white tank over the white half. Results indicated that the tank wall color significantly affected depth preference, with the transparent tank producing the strongest depth preference and the black tank producing the weakest preference. Tank depth, however, did not significantly affect color preference. Additionally, wall color significantly affected shuttling and immobility, while depth significantly affected shuttling and thigmotaxis. These results are consistent with previous indications that the diving response and scototaxis may reflect dissociable mechanisms of behavior. We conclude that the two tests are complementary rather than interchangeable, and that further research on the motivational systems underlying behavior in each of the two tests is needed.     

Short-term and long-term habituation of exploration in rats,hamsters and gerbils

Short-term (within-session) and long-term (between-session) habituation of exploratory behavior was studied in rats, hamsters and gerbils. Subjects were observed in an open field containing four different objects during three 15-min sessions with an 8–14 h interval between each session. Their exploratory activity was measured by the numbers of contacts they made with these objects. The three species differed from each other in both their long-term and short-term habituation of exploration. Rats showed disrupted between-session habituation because of an important initial burst of activity at the beginning of each session. In contrast, hamsters and gerbils displayed between-session habituation, but within-session habituation occured during the first session only. These results are discussed in relation both to the adaptive value of exploration, and to the natural habitat of each species.     

The circadian Clock mutation increases exploratory activity and escape-seeking behavior

Disturbances of circadian rhythms are associated with many types of mood disorders; however, it is unknown whether a dysfunctional circadian pacemaker can be the primary cause of altered emotional behavior. To test this hypothesis, male and female mice carrying a mutation of the circadian gene, Clock, were compared to wild-type mice in an array of behavioral tests used to measure exploratory activity, anxiety, and behavioral despair. Female Clock mutant mice exhibited significantly greater activity and rearing in an open field and a greater number of total arm entries in the elevated plus maze. In addition, female Clock mutant mice spent significantly more time swimming in the forced swim test than wild-type mice on both days of a 2-day test. Male Clock mutant mice also exhibited increased exploration of the open field and increased swimming in the forced swim test; however, behavioral changes were less robust in Clock mutant males compared to Clock mutant females. These changes in behavior were not dependent on the expression of a lengthened free-running period but were more or less striking depending on the testing conditions. These data indicate that the Clock mutation leads to increased exploratory behavior and increased escape-seeking behavior, and, conversely, does not result in increased anxiety or depressive-like behavior. These results suggest that the Clock gene is involved in regulating behavioral arousal, and that Clock may interact with sex hormones to produce these behavioral changes.     

Mefenamic Acid Attenuates Chronic Alcohol Induced Cognitive Impairment in Zebrafish: Possible Role of Cholinergic Pathway

Based on the scientific evidence supporting the neuroinflammatory response contributes the cognitive impairment associated with chronic alcoholism and the neuroprotective actions of mefenamic acid with reversal of memory loss and brain inflammation in mice, this study was designed to evaluate the effect of mefenamic acid against chronic alcohol induced cognitive impairment in zebrafish model. Zebrafish were grouped and subjected to normal behavioral analysis in light–dark chamber for 10 days. The preference to dark compartment was noted in zebrafish. Zebrafish were grouped and exposed to escalating doses of alcohol for 28 days with and without mefenamic acid exposure (100 and 200 µg/L) and subjected to a fear conditioning passive avoidance task from day 13 of 28. The cognitive evaluation was performed for 10 days and the brain tissue was isolated to estimate acetylcholinesterase activity. In cognitive evaluation study, the normal zebrafish retained the memory of the learned task and avoided the dark. The alcohol exposed zebrafish showed impairment in retaining the memory of learned task. Mefenamic acid exposed zebrafish showed a significant protection against cognitive impairment caused by alcohol and retained the memory of learned task with a significant decrease in AChE activity in brain homogenate compared to alcohol exposed zebrafish. The results of this study suggest that the memory enhancing activity of mefenamic acid might be due to activation of cholinergic transmission that has protected neuroinflammatory and neurodegenerative conditions caused by alcohol.     

Moniliformis moniliformis infection has no effect on some behaviors of the cockroach Diploptera punctata.

The behavior of the cockroach Diploptera punctata parasitized with the acanthocephalan Moniliformis moniliformis was examined for parasite-induced alterations. No significant difference in behavior was found between parasitized and unparasitized animals in the following behavioral tests: (1) choice of white/black, horizontal/vertical substrate under light and dark conditions; (2) temporal and directional response to a bright light source; (3) choice between light and dark (photophilia); and (4) activity (time spent moving, distance, and velocity). A comparison of uninfected animals under 2 light conditions showed that light affected the activity of uninfected animals and their response to substrate. Diploptera punctata is the first nondomestic cockroach to be examined for behavioral responses to Moniliformis infection. This is the first report of an arthropod in which acanthocephalan infection has failed to alter behavior under at least some common test conditions.