This work was supported by &#34;Agencia Estatal de Investigaci&#243;n&#34; from the Spanish Ministry of Science and Innovation (project PID2020-113371RB-C21), IDAEA-CSIC, Severo Ochoa Centre of Excellence (CEX2018-000794-S). Juliette Bedrossiantz was supported by a PhD grant (PRE2018-083513) co-financed by the Spanish Government and the European Social Fund (ESF).

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

<ol>
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M., Kalueff, A., Cachat, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-Aided+Analysis+of+Zebrafish+Locomotion+and+Anxiety-Related+Behavioral+Responses.">Video-Aided Analysis of Zebrafish Locomotion and Anxiety-Related Behavioral Responses.</a> Zebrafish Neurobehavioral Protocols. Neuromethods. 51, (2011).</li><li>Rosemberg, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+spatio-temporal+behavior+of+zebrafish+in+the+open+tank+paradigm+after+a+short-period+confinement+into+dark+and+bright+environments.">Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments.</a> PLoS ONE. 6 (5), e19397 (2011).</li><li>Blaser, R., Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+in+Zebrafish:+Comparison+of+three+behavioral+quantification+methods.">Behavioral phenotyping in Zebrafish: Comparison of three behavioral quantification methods.</a> Behavioral Research Methods. 38 (3), 456-469 (2006).</li><li>Cachat, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+neurophenotyping+of+adult+zebrafish+behavior.">Three-dimensional neurophenotyping of adult zebrafish behavior.</a> PLoS ONE. 6 (3), e17597 (2011).</li><li>Cachat, J. 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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Characteristic anxiety behaviors observed in NTT have been positively correlated with serotonin levels analyzed in brains21. For example, after exposure to para-chlorophenylalanine (PCPA), an inhibitor of 5-HT biosynthesis, fish exhibited positive geotaxis as well as decreased brain 5-HT levels22, results very similar to those obtained with METH. Therefore, the decrease in brain serotonin levels and the display of positive geotaxis in METH-exposed zebrafish suggests that the anxiety behavior produced by the drug is mediated by the serotonergic pathway. Interestingly, a similar behavioral phenotype, i.e., an anxiogenic effect on geotaxis, can be seen in adult zebrafish exposed for 2 weeks to 0.3 3 &#181;g/L and 3 &#181;g/L, two environmentally relevant concentrations of glyphosate. An increase in geotaxis was also previously reported for adult zebrafish with the neurotoxicant acrylamide6,23. In all these cases, this behavioral phenotype (an increase of geotaxis in the NTT, characteristic of anxiogenic substance) was associated with the diminution of monoaminergic neurotransmitter levels. Therefore, the NTT paradigm combined with neurochemical analysis of the brain provides ecologically relevant information, exploratory behavior, and foraging efficiency and connects behavior neurophenotypes with neurotransmitter modulations. 
On the other hand, an impairment of social behaviors in both assays, the ST and SPT, was also observed in METH-treated fish. The result obtained in this study is consistent with several studies with rats and monkeys, where the acute and chronic exposure of the study animals to METH results in social withdrawal24. Social behavior changes associated with METH abuse have been explained in humans by impairments in social-cognitive function24. An anxiogenic effect on the shoal size was found in zebrafish exposed for 2 weeks to 3 &#181;g/L glyphosate. We observed a phenocopy of this effect in zebrafish exposed to 53 mg/L (0.75 mM) acrylamide for 3 days6,23. 
The NTT, ST, and SPT assays allow to effectively determine the potential neurotoxic effects25 of a wide range of chemicals as illustrated by the study of acute methamphetamine and sub-chronic glyphosate toxicity models in adult zebrafish. Behavior is, in toxicology, a relevant apical endpoint, characterizing the effects at organismal levels of a chemical for neurotoxicity and environmental research. Besides being a sublethal endpoint in laboratory conditions, changes in behaviors, such as exploratory or social behavior, can be deleterious in nature. Moreover, the proposed behavioral analysis battery is an easy-to-implement, semi-automated method11 and, therefore, very efficient if the assays are consciously planned (reduction principle)26. Performing these assays as a test battery using a single tank reduces the number of animals and the experimental time and waste generation. 
The order of the assays in the battery is an important consideration if we want to study the response profile of an individual in each trial. For this purpose, conducting the individual assays followed (see Figure 2) allows for keeping the animal identified and to relate its exploratory behavior to its social preference. In addition, the animal's behavioral responses can be related to other biological data, such as its neurotransmitter profile or gene expression, if the fish are kept identified until the end point of sampling (Figure 2A). 
Usually, behavioral analysis allows for the observation of differences between groups. First, individual responses are calculated on the basis of animal tracking27 before pooling the data by group. Then, the means and the difference in variance with respect to the control group are compared for each behavioral parameter calculated. With shoaling analysis12, it is critical to be very clear that the unit of variance is the group of test fish, not individual fish because the behavior of each individual fish is influenced by the other fish in the shoal. This is the way used in most papers to process behavioral data28. However, it might be useful to rethink the analysis of behavioral parameters not on a parameter-by-parameter basis but as an overall response per trial. For example, one could calculate the covariance of each measurement made in a trial and report it as a different way of measuring the same thing: anxious, exploratory, or gregarious behavior. There are many ways to calculate and interpret behavioral data28,29. Depending on the number of conditions, type of tests, and image acquisition (2D or 3D)30,31 the analysis can be completely rethought in order to get the best out of the data.

The zebrafish (Danio rerio) is a popular model vertebrate species for ecotoxicology, drug discovery, and safety pharmacology studies. Its low cost, well-established molecular genetic tools, and conservation of key physiological processes involved in the morphogenesis and maintenance of the nervous system make zebrafish an ideal animal model for neuroscience research, including neurobehavioral toxicology1,2. The main endpoint for evaluating the neurotoxicity of a chemical was, until recently, the presence of neuropathological effects. Lately, however, behavioral endpoints have been incorporated into neurotoxicological screening protocols, and these functional outcomes are now commonly used to identify and determine the potential neurotoxicity of chemicals3,4. Moreover, behavioral endpoints are highly relevant from an ecological point of view, as even a very mild behavioral change in fish could endanger the survival of the animal in natural conditions5.
One of the most used behavioral assays in adult zebrafish research is the novel tank test (NTT), which measures anxiety-like behavior6,7. In this assay, fish are exposed to novelty (fish are placed in an unfamiliar tank), a mild aversive stimulus and their behavioral responses are observed. NTT is used to assess basal locomotor activity, geotaxis, freezing, and erratic movements of fish, principally. Erratic8 is characterized by abrupt changes of direction (zigzagging) and repeated episodes of accelerations (darting). It is an alarm reaction and is usually observed before or after freezing episodes. Freezing behavior corresponds to a complete cessation of the fish&#39;s movements (except for opercular and ocular movements) while on the bottom of the tank, as distinguished from immobility caused by sedation, which causes hypolocomotion, akinesia, and sinking8. Freezing is usually related to a high state of stress and anxiety and is also part of submissive behavior. Complex behaviors are excellent indicators of the state of anxiety of animals. NTT has been shown to be sensitive to pharmacological and genetic manipulation9, making it a valuable tool for studying the neural basis of anxiety and related disorders.
Zebrafish are a highly social species, so we can measure a wide range of social behaviors. The shoaling test (ST) and the social preference test (SPT) are the most used assays to assess social behavior10. The ST measures the tendency of fish to group together11 by quantifying their spatial behavior and movement patterns. ST is useful for studying group dynamics, leadership, social learning, and understanding the social behavior of many fish species12. The SPT in adult zebrafish was adapted from Crawley&#39;s preference for social novelty test for mice13&#160;and quickly became a popular behavioral assay for the study of social interaction in this model species14. These two tests have also been adapted for use in drug screening assays and have shown promise for identifying novel compounds that modulate social behavior15,16.
In general, behavioral assays in adult zebrafish are powerful tools that can provide valuable information on the behavior mechanisms or the neurophenotypes of active compounds and abused drugs17. This protocol details how to implement these behavioral tools7 with basic material resources and how to apply them in toxicity assays to characterize the effects of a wide range of neuroactive compounds. In addition, we will see that the same tests can be applied to assess the neurobehavioral effects of acute exposure to a neuroactive compound (methamphetamine) but also to characterize these effects after chronic exposure to environmental concentrations of a pesticide (glyphosate).

<table><tbody><tr><td>Aquarium Cube shape</td><td>Blau Aquaristic</td><td>7782025</td><td>Cubic Panoramic 10 &amp;nbsp;(10 L, 20 cm x 20 cm x 25 cm, 5 mm)</td></tr><tr><td>Ethovision software</td><td>Noldus</td><td>Ethovision XT</td><td>Version 12.0 or newer</td></tr><tr><td>GigE camera</td><td>Imaging Development Systems</td><td>UI-5240CP-NIR-GL</td><td></td></tr><tr><td>GraphPad Prism 9.02</td><td>GraphPad software Inc</td><td>GraphPad Prism 9.02</td><td>&amp;nbsp;For Windows</td></tr><tr><td>IDS camera manager</td><td>Imaging Development Systems</td><td></td><td></td></tr><tr><td>LED backlight illumination</td><td>Quirumed</td><td>GP-G2</td><td></td></tr><tr><td>SPSS Software</td><td>IBM</td><td>IBM SPSS v26</td><td></td></tr><tr><td>uEye Cockpit software&amp;nbsp;</td><td>Imaging Development Systems</td><td>version 4.90</td><td></td></tr></tbody></table>

neurotoxicity assessment in adult danio rerio using a battery of behavioral tests in a single tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

In this section, we will look at some possible applications of these behavioral tools in fish neurotoxicology. The following results correspond to the characterization of the acute or binge effects of methamphetamine (METH), a recreational drug, and the sub-chronic effects of glyphosate, one of the main herbicides found in aquatic ecosystems.
Characterization of a methamphetamine binge neurotoxicity model in adult zebrafish 
When evaluating the effect of 40 mg/L METH on NTT (Figure 3), the Kruskal-Wallis test confirmed that the exposed animals presented a positive geotaxis, characterized by a decrease in the exploration time in the upper zone of the experimental tank (H(2) = 35.964, P = 1.55 x 10-8), as well as in the distance traveled in this part (H(2) = 32.272, P = 9.82 x 10-8), and in the number of visits (H(2) = 36.527, P = 1.17 x 10-8). We also observed a significant increase in the latency time preceding the first visit to the upper zone (H(2) = 17.264, P = 0.00018). It is important to remark that the differences observed in the parameters measured in the NTT after METH exposure are consistent over time, as confirmed by the Bonferroni correction (P &#62; 0.8). A significant effect of exposure time was found for freezing behavior (H(2) = 13.120, P = 0.0014).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65869/65869fig03.jpg" /> 
Figure 3: Anxiety-like behavior assessed in standard 6-min novel tank test (NTT) of adult zebrafish exposed to 40 mg/L methamphetamine (METH) for 3 h and 48 h. Data from each experiment were normalized to the corresponding control values. The combined data is reported as a scatter plot with the median (n = 14-15), **p &#60; 0.01, ***p &#60; 0.001; Kruskal Wallis test with Bonferroni correction for NTT endpoints. Data from 2 independent experiments. This figure has been reproduced with permission from Bedrossiantz et al.15. <a href="https://www.jove.com/files/ftp_upload/65869/65869fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Freezing movements can be quantified by assessing the frequency, latency, duration, or location of freezing. The best way to score them is undoubtedly the eye of an experienced observer, which is quite laborious and complex, so we tried an automated alternative using EthoVision software to detect freezing behavior19. We found that the number, latency, and duration of freezing attacks calculated by the software (Table 1A) correlate with good accuracy with the episodes scored manually by the observer (Table 1B). Whereas the two methods are equivalent in terms of results (P = 0.958, Student&#39;s test), we used the automated approach to assess the freezing here. After 3 h of exposure to METH, freezing time increased significantly (P = 0.0012), whereas no difference was found with the control after 48 h of exposure (P = 0.16). METH produced no effect on erratic movements at either time.
We used two experimental paradigms to evaluate the effects on social behavior after acute exposure to METH. The ST (Figure 4) revealed that the average distance and farthest distance between individuals were significantly greater for METH-treated fish (H(2) = 53.261, P = 2.72 x 10-12; H(2)=52.504, P = 3.97 x 10-12 for average and farthest interfish distances, respectively), pointing to a behavioral phenotype of social isolation. Again, we remark that no time effect was found using the Bonferroni post hoc test (P &#62; 0.5).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65869/65869fig04.jpg" /> 
Figure 4: Social behavior of adult zebrafish waterborne exposed to 40 mg/L methamphetamine (METH) for 3 h and 48 h. Shoaling test (ST) results, including the average and the farthest interfish distances. The combined data is reported as a scatter plot with the median (n = 18), *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001; Kruskal Wallis test with Bonferroni correction. Data from 2 independent experiments. This figure has been reproduced with permission from Bedrossiantz et al.15. <a href="https://www.jove.com/files/ftp_upload/65869/65869fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In the SPT (Figure 5), treated fish show a significant decrease in time spent and distance traveled in the conspecific zone (F(2,74) = 14.497, P = 4.87 x 10-6; F(2,73) = 13.461, P = 0.00001 for time spent and distance traveled in the conspecific zone, respectively). These results reaffirm the social isolation phenotype suggested by the TS results. Tukey&#39;s Honest Significant Difference (HSD) post hoc test ruled out no possible differences between the two analysis times (P &#62; 0.5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65869/65869fig05.jpg" /> 
Figure 5: Social behavior of adult zebrafish waterborne exposed to 40 mg/L methamphetamine (METH) for 3 h and 48 h. The social preference test (SPT) results, including time and distance of the fish in each of the three virtual zones of the experimental tank: empty, center, and conspecific. Data from each experiment were normalized to the corresponding control values. The combined data is reported as a scatter plot with the median (n = 17-20), *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001; one-way ANOVA with Dunnett&#39;s multiple comparison test. Data from 2 independent experiments. This figure has been reproduced with permission from Bedrossiantz et al.15. <a href="https://www.jove.com/files/ftp_upload/65869/65869fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Behavioral effect of sub-chronic exposure to environmental levels of glyphosate 
Behavioral analysis of the effects of sub-chronic exposure to 3 &#181;g/L glyphosate on the NTT (Figure 6) reveals a significant decrease in time spent exploring the top (F2,77 = 8.744, P = 0.0004), distance traveled in this part (F2,77 = 9.118, P = 0.0003), and number of visits (F2,77 = 3.441, P = 0.037). These effects are characteristic of positive geotaxis behavior, as is the increased effect observed on the latency time preceding the first visit to the top of the tank (H(2) = 9.628, P = 0.008). The expression of erratic and freezing behaviors of the exposed animals was also analyzed in the NTT. The duration (H(2) = 17.261, P = 0.025) and number of erratic episodes (F2,76 = 10.073, P = 0.0001) were significantly increased by glyphosate. In contrast, no freezing differences were found with the control (Pearson Chi-Square(2) = 2.964, P = 0.253). Applied to an ecological context, the observations made at NTT suggest that glyphosate could significantly decrease the exploratory behavior of fish, jeopardizing their ability to survive in the wild.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65869/65869fig06.jpg" /> 
Figure 6: Anxiety-like behavior assessed in standard 6-min novel tank test (NTT) of adult zebrafish exposed to 0.3 &#181;g/L and 3 &#181;g/L glyphosate for 2 weeks. Behavioral parameters analyzed, as well as a cartoon of the experimental tank divided into two equal virtual zones, top and bottom. Data reported as a scatter plot with the median (n = 23-29), *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001; one-way ANOVA with Dunnett&#39;s multiple comparison test (Total distance, Distance in top, Time in top, Transitions to top, Erratic bouts, High mobility frequency) or Kruskal Wallis test with Bonferroni correction (Latency to the top, Erratic duration). No differences (P &#62; 0.05) were found in the freezing duration and freezing bouts. Data from 2-4 independent experiments. This figure has been reproduced with permission from Faria et al.20. <a href="https://www.jove.com/files/ftp_upload/65869/65869fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Schooling, non-polarized groups of conspecifics that are held together by social pressure to protect themselves from predators, is a natural tendency of Danio rerio. The school can &#34;tighten&#34; or &#34;expand&#34; depending on the animals&#39; level of anxiety or fear, a particular visual effect that is very easy to identify experimentally (Figure 7). In the glyphosate experiment, the shoaling test revealed an increase in anxiety in fish exposed to 3 &#181;g/L, reflected by a grouping of the shoal and thus a significant decrease in the average distance and farthest distance between individuals (F2,56 = 5.664, P = 0.006 and F2,56 = 7.413, P = 0.001, for the average and farthest interfish distances, respectively) compared to the control.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65869/65869fig07.jpg" /> 
Figure 7: Social behavior of adult zebrafish waterborne exposed to 0.3 &#181;g/L and 3 &#181;g/L glyphosate for 2 weeks. Data reported as scatter plot with the median (n = 19-20), *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001; one-way ANOVA with Dunnett&#39;s multiple comparison test (Average interfish distance and Farthest distance) Data from 2 to 4 independent experiments. This figure has been reproduced with permission from Faria et al.20. <a href="https://www.jove.com/files/ftp_upload/65869/65869fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: An approximation of freezing behavior using an automated analysis. Data reported in this table come from the same recording (Video 1) analyzed with two different methods. (A) Approximation of freezing behavior by automated calculation with EthoVision V13 software. The variable mobility is calculated from the change of the subject area between two samples, so it depends on the acquisition frequency of this area. We set a very low threshold of immobility (less than 3% mobility) as well as the sample rate to a minimum continuous time of 5 s (more than 150 frames). (B) Analysis of freezing behavior with Behavioral Observation Research Interactive Software (BORIS, free and open-source software). BORIS is an event-logging software for video coding and live observations. With BORIS, the observer can code the freeze episode as a state event, defining the start and end points. <a href="https://www.jove.com/files/ftp_upload/65869/Table 1.xls" target="_blank">Please click here to download this Table.</a>
Video 1: Control fish in the novel tank test. <a href="https://www.jove.com/files/ftp_upload/65869/JoVE [C10 24h]_Video 1.zip" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish at JoVE.com

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

neurotoxicity-assessment-adult-danio-rerio-using-battery-behavioral

Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.6K Views.  Institute for Environmental Assessment and Water Research (IDAEA-CSIC). Ons belangrijkste onderzoek is gericht op het identificeren van de gedragseffecten die worden veroorzaakt door milieuverontreinigende stoffen en medicijnen bij vissen. Ook proberen we de mechanismen te begrijpen die tot deze effecten leiden. Een sociaal en angstachtig gedrag kan niet gemakkelijk worden aangepakt met behulp van embryo's.We voeren studies uit die gericht zijn op dit gedrag in het volwassen zebravismodel. Dit protocol beschrijft hoe gedragsinstrumenten kunnen worden geïm...

Video: Auteur Spotlight: Beoordeling van milieu- en door drugs geïnduceerde gedragsveranderingen bij volwassen zebravissen 

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Environment

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Behavioral Approaches to Studying Innate Stress in Zebrafish

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of ...

Behavior

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that can restore these neurons. Much effort is currently directed toward a better understanding of neuroregeneration using preclinical in vivo models. This regenerative capability for self-repair is, however, inefficient in mammals. Non-mammalian animals like zebrafish have thus emerged as an excellent neuroregenerative model due to its capability to continuously self-renew and have a close brain homology to humans. As part of the effort in elucidating cellular events involved in neuroregeneration in vivo, we have established the 6-hydroxydopamine (6-OHDA)-induced adult zebrafish-based PD model. This was achieved through the optimized intracerebroventricular (ICV) microinjection of 99.96&#160;mM 6-OHDA to specifically ablate dopaminergic neurons (DpN) in the ventral diencephalon (Dn) of zebrafish brain. Immunofluorescence indicated more than 85% of DpN ablation at day three postlesion and full restoration of DpN at lesioned site 30 days postlesion. The present study determined the impairment and subsequent recovery of zebrafish swimming behavior following lesion by using the open field test through which two parameters, distance traveled (cm) and mean speed (cm/s), were quantified. The locomotion was assessed by analyzing the recordings of individual fish of each group (n = 6) using video tracking software. The findings showed a significant (p &#60; 0.0001) reduction in speed (cm/s) and distance traveled (cm) of lesioned zebrafish 3 days postlesion when compared to sham. The lesioned zebrafish exhibited full recovery of swimming behavior 30 days postlesion. The present findings suggest that 6-OHDA lesioned adult zebrafish is an excellent model with reproducible quality to facilitate the study of neuroregeneration in PD. Future studies on the mechanisms underlying neuroregeneration as well as intrinsic and extrinsic factors that modulate the process may provide important insight into new cell replacement treatment strategies against PD.

The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking software.

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that ...

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human health. This deficit of chemical safety information is exacerbated by current testing methods in mammals that are expensive, labor-intensive, and time-consuming. Recently, scientists and regulators have been working to develop new approach methodologies (NAMs) for chemical safety testing that are cheaper, more rapid, and reduce animal suffering. One of the key NAMs to emerge is the use of invertebrate organisms as replacements for mammalian models to elucidate conserved chemical modes of action across distantly related species, including humans. To advance these efforts, here, we describe a method that uses the fruit fly, Drosophila melanogaster,&#160;to assess chemical safety. The protocol describes a simple, rapid, and inexpensive procedure to measure the viability and feeding behavior of exposed adult flies. In addition, the protocol can be easily adapted to generate samples for genomic and metabolomic approaches. Overall, the protocol represents an important step forward in establishing Drosophila as a standard model for use in precision toxicology.

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster.

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human ...

Biology

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

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