The authors have no acknowledgements.

<ol>
	<li>Brennan, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+behavioural+assays+of+translational+relevance+for+the+study+of+psychiatric+disease.">Zebrafish behavioural assays of translational relevance for the study of psychiatric disease.</a> Rev. Neurosci. 22, 37-48 (2011).</li><li>Kabashi, E., Brustein, E., Champagne, N., Drapeau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+for+the+functional+genomics+of+neurogenetic+disorders.">Zebrafish models for the functional genomics of neurogenetic disorders.</a> Biochim. Biophys. Acta. 1812, 335-345 (2011).</li><li>McCollum, C. W., Ducharme, N. A., Bondesson, M., Gustafsson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+screening+in+zebrafish.">Developmental toxicity screening in zebrafish.</a> Birth. Defects. Res. C. Embryo Today. 93, 67-114 (2011).</li><li>Kurta, A., Palestis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ethanol+on+the+shoaling+behavior+of+zebrafish+(Danio+rerio).">Effects of ethanol on the shoaling behavior of zebrafish (Danio rerio).</a> Dose-Response. 8, 527-533 (2010).</li><li>Sison, M., 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+performance+altering+effects+of+MK-801+in+zebrafish+(Danio+rerio).">Behavioral performance altering effects of MK-801 in zebrafish (Danio rerio).</a> Behav. Brain Res. 220, 331-337 (2011).</li><li>Delcourt, J., Deno&#235;l, M., Ylieff, M., Poncin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video+multitracking+of+fish+behaviour:+a+synthesis+and+future+perspectives.">Video multitracking of fish behaviour: a synthesis and future perspectives.</a> Fish Fish. 14, 186-204 (2013).</li><li>Zhu, L., Weng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catadioptric+stereo-vision+system+for+the+real-time+monitoring+of+3D+behavior+in+aquatic+animals.">Catadioptric stereo-vision system for the real-time monitoring of 3D behavior in aquatic animals.</a> Physiol. Behav. 91, 106-119 (2007).</li><li>Zhu, L., Wang, J., Su, R., Weng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System+for+3D+monitoring+and+analysis+of+motion+behavior+of+targets.">System for 3D monitoring and analysis of motion behavior of targets.</a> US patent. , (2012).</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, e17597 (2011).</li><li>Maaswinkel, H., Zhu, L., Weng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immediate+and+the+delayed+effects+of+buspirone+on+zebrafish+(Danio+rerio)+in+an+open+field+test:+a+3-D+approach.">The immediate and the delayed effects of buspirone on zebrafish (Danio rerio) in an open field test: a 3-D approach.</a> Behav. Brain Res. 234, 365-374 (2012).</li><li>Maaswinkel, H., Le, X., He, L., Zhu, L., Weng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociating+the+effects+of+habituation,+black+walls,+buspirone+and+ethanol+on+anxiety-like+behavioral+responses+in+shoaling+zebrafish.+A+3D+approach+to+social+behavior.">Dissociating the effects of habituation, black walls, buspirone and ethanol on anxiety-like behavioral responses in shoaling zebrafish. A 3D approach to social behavior.</a> Pharmacol. Biochem. Behav. 108, 16-27 (2013).</li><li>Maaswinkel, H., Zhu, L., Weng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advantages+of+a+3D-automatic+recording+system+for+the+study+of+shoaling+behavior+in+zebrafish.">The advantages of a 3D-automatic recording system for the study of shoaling behavior in zebrafish.</a> , (2012).</li><li>Maaswinkel, H., Zhu, L., Weng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automatic+recording+system+to+quantify+antagonistic+interactions+between+zebrafish.+Abstract.">An automatic recording system to quantify antagonistic interactions between zebrafish. Abstract.</a> , (2012).</li><li>Spence, R., Gerlach, G., Lawrence, C., Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+and+ecology+of+the+zebrafish,+Danio+rerio.">The behaviour and ecology of the zebrafish, Danio rerio.</a> Biol. Rev. Camb. Phil. Soc. 83, 13-34 (2008).</li><li>Krause, J., Hartmann, N., Pritchard, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+nutritional+state+on+shoal+choice+in+zebrafish,+Danio+rerio.">The influence of nutritional state on shoal choice in zebrafish, Danio rerio.</a> Anim. Behav. 57, 771-775 (1999).</li><li>Speedie, N., 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=Alarm+substance+induced+behavioral+responses+in+zebrafish+(Danio+rerio).">Alarm substance induced behavioral responses in zebrafish (Danio rerio).</a> Behav. Brain Res. 188, 168-177 (2008).</li><li>Gebauer, D. L., Pagnussat, N., Piato, A. L., Schaeffer, I. C., Bonan, C. D., Lara, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+anxiolytics+in+zebrafish:+similarities+and+differences+between+benzodiazepines,+buspirone+and.">Effects of anxiolytics in zebrafish: similarities and differences between benzodiazepines, buspirone and.</a> 99, 480-486 (2011).</li><li>Paull, G. C., Filby, A. L., Giddins, H. G., Coe, T. S., Hamilton, P. B., Tyler, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominance+hierarchies+in+zebrafish+(Danio+rerio)+and+their+relationship+with+reproductive+success.">Dominance hierarchies in zebrafish (Danio rerio) and their relationship with reproductive success.</a> Zebrafish. 7, 109-117 (2010).</li><li>Engeszer, R. E., Ryan, M. J., Parichy, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learned+social+preference+in+zebrafish.">Learned social preference in zebrafish.</a> Curr. Biol. 14, 881-884 (2004).</li><li>Mathur, P., Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+zebrafish+as+a+model+to+understand+mechanisms+of+addiction+and+complex+neurobehavioral+phenotypes.">Use of zebrafish as a model to understand mechanisms of addiction and complex neurobehavioral phenotypes.</a> Neurobiol. Dis. 40, 66-72 (2010).</li><li>Tropepe, V., Sive, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+zebrafish+be+used+as+a+model+to+study+the+neurodevelopmental+causes+of+autism.">Can zebrafish be used as a model to study the neurodevelopmental causes of autism.</a> Genes Brain Behav. 2, 268-281 (2003).</li><li>Echevarria, D. J., Hammack, C. M., Pratt, D. W., Hosemann, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+behavioral+test+battery+to+assess+global+drug+effects+using+zebrafish.">A novel behavioral test battery to assess global drug effects using zebrafish.</a> Int. J. Comp. Psychol. 21, 19-34 (2008).</li><li>Seibt, K. J., Piato, A. L., Luz Oliveira, d. a., Capiotti, R., Vianna, K. M., R, M., Bonan, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antipsychotic+drugs+reverse+MK-801+induced+cognitive+and+social+interaction+deficits+in+zebrafish+(Danio+rerio).">Antipsychotic drugs reverse MK-801 induced cognitive and social interaction deficits in zebrafish (Danio rerio).</a> Behav. Brain. Res. 224, 135-139 (2011).</li><li>Zou, H., 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=Low+dose+MK-801+reduces+social+investigation+in+mice.">Low dose MK-801 reduces social investigation in mice.</a> Pharmacol. Biochem. Behav. 90, 753-757 (2008).</li><li>Ng, M. C., Hsu, C. P., Wu, Y. J., Wu, S. Y., Yang, Y. L., Lu, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+MK-801-induced+impairment+of+inhibitory+avoidance+learning+in+zebrafish+via+inactivation+of+extracellular+signal-regulated+kinase+(ERK)+in+telencephalon.">Effect of MK-801-induced impairment of inhibitory avoidance learning in zebrafish via inactivation of extracellular signal-regulated kinase (ERK) in telencephalon.</a> Fish Physiol. Biochem. 38, 1099-1106 (2012).</li><li>Sison, M., 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=Associative+learning+performance+is+impaired+in+zebrafish+(Danio+rerio)+by+the+NMDA-R+antagonist+MK-801.">Associative learning performance is impaired in zebrafish (Danio rerio) by the NMDA-R antagonist MK-801.</a> Neurobiol. Learn. Mem. 96, 230-237 (2011).</li><li>Swain, H. A., Sigstad, C., Scalzo, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dizocilpine+(MK-801)+on+circling+behavior,+swimming+activity,+and+place+preference+in+zebrafish+(Danio+rerio).">Effects of dizocilpine (MK-801) on circling behavior, swimming activity, and place preference in zebrafish (Danio rerio).</a> Neurotox. Teratol. 26, 725-729 (2004).</li><li>Vezzani, A., 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=Kinetics+of+MK-801+and+its+effect+on+quinolinic+acid-induced+seizures+and+neurotoxicity+in+rats.">Kinetics of MK-801 and its effect on quinolinic acid-induced seizures and neurotoxicity in rats.</a> J. Pharmacol. Exp. Ther. 249, 278-283 (1989).</li><li>Green, 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=Automated+high-throughput+neurophenotyping+of+zebrafish+social+behavior.">Automated high-throughput neurophenotyping of zebrafish social behavior.</a> J. Neurosci. Methods. 210, 266-271 (2012).</li><li>Rung, J. P., Carlsson, A., Ryd&#233;n Markinhuhta, K., Carlsson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+(+)-MK-801+induced+social+withdrawal+in+rats;+a+model+for+negative+symptoms+of+schizophrenia."> (+)-MK-801 induced social withdrawal in rats; a model for negative symptoms of schizophrenia.</a> Prog. Neuro-psychopharmacol. Biol. Psych. 29, 827-832 (2005).</li><li>Gururajan, A., Taylor, D. A., Malone, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+and+clozapine+reverse+MK-801-induced+deficits+in+social+interaction+and+hyperactivity+in+Sprague-Dawley+rats.">Cannabidiol and clozapine reverse MK-801-induced deficits in social interaction and hyperactivity in Sprague-Dawley rats.</a> J. Psychopharmacol. 226, 1317-1332 (2012).</li><li>Burket, J. A., Cannon, W. R., Jacome, L. F., Deutsch, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MK-801,+a+noncompetitive+NMDA+receptor+antagonist,+elicits+circling+behavior+in+the+genetically+inbred+Balb/c+mouse+strain.">MK-801, a noncompetitive NMDA receptor antagonist, elicits circling behavior in the genetically inbred Balb/c mouse strain.</a> Brain Res. Bull. 83, 337-339 (2010).</li><li>Saunders, J. A., Gandal, M. J., Roberts, T. P., Siegel, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+antagonist+MK801+recreates+auditory+electrophysiology+disruption+present+in+autism+and+other+neurodevelopmental+disorders.">NMDA antagonist MK801 recreates auditory electrophysiology disruption present in autism and other neurodevelopmental disorders.</a> Behav. Brain Res. 234, 233-237 (2012).</li><li>Brosda, 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=Pharmacological+and+parametrical+investigation+of+prepulse+inhibition+of+startle+and+prepulse+elicited+reactions+in+Wistar+rats.">Pharmacological and parametrical investigation of prepulse inhibition of startle and prepulse elicited reactions in Wistar rats.</a> Pharmacol. Biochem. Behav. 99, 22-28 (2011).</li><li>Wong, K., 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=Analyzing+habituation+responses+to+novelty+in+zebrafish+(Danio+rerio).">Analyzing habituation responses to novelty in zebrafish (Danio rerio).</a> Behav. Brain Res. 208, 450-457 (2010).</li><li>Maximino, C., 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=Parametric+analyses+of+anxiety+in+zebrafish+scototaxis.">Parametric analyses of anxiety in zebrafish scototaxis.</a> Behav. Brain Res. 210, 1-7 (2010).</li><li>Blaser, R. E., Rosemberg, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+anxiety+in+zebrafish+(Danio+rerio):+dissociation+of+black/white+preference+and+novel+tank+test.">Measures of anxiety in zebrafish (Danio rerio): dissociation of black/white preference and novel tank test.</a> PloS One. 7, e36931 (2012).</li></ol>

Hans Maaswinkel, Liqun Zhu and Wei Weng are employees of xyZfish, Ronkonkoma NY, that produces the tracking system used in this article.

The experimental procedure shown here allows recording individual zebrafish, pairs of zebrafish (e.g. to study mating or aggression) and shoals of zebrafish (as shown in the representative example). The system is especially suited for simultaneously obtaining information about 3D (vertical and horizontal) distribution, kinematic characteristics (e.g. velocity, acceleration, turning angle) and social parameters (e.g. social cohesion). In the following, we list a few important issues to consider when planning experiments.
Depending on the experimental procedure, different behavioral systems can be studied. For instance, to explore neophobia (i.e. fear for new environments) the recording should be started immediately after introducing the zebrafish to the observation tank (after waiting just for a few seconds for the water to settle down to diminish noise), without prior habituation11,17,37. On the other hand, some research topics might require the zebrafish to be well habituated, for instance when studying the effects of alarm pheromones16. When studying groups of zebrafish, the experimenter should be aware that although the software minimizes switching of tags, it is at this moment not possible to follow individual zebrafish throughout. This is also not important for shoaling studies where the emphasis is on social cohesion, but would be crucial when exploring aggression or mating. Manual reassignment of tags is an option, but can be very time-consuming.
The color of the walls of the observation container and of the observation chamber can have great influence on the behavior of the zebrafish. For instance, white walls have been described to induce aversion responses36, 37. When trying different colors, keep two things in mind: 1) it is important that the front wall (i.e. the wall closest to the camera) remains transparent and that a lid (if one is used) also has to be transparent; 2) when selecting a color never used before, first determine whether the contrast between zebrafish and background is appropriate for recording. Do a test run. Changing the camera settings for recording (Gain, Shutter, Red and Blue, as explained in steps 2.5 and 2.6) and the settings for trajectory reconstruction (e.g. color and size thresholds, as explained in steps 3.3, 3.5, 3.11, and 3.12) can make a big difference. Again, experience with the system will go a long way to decide what is possible.
Depending on the purpose of the experiment, a light source with different characteristics (e.g. spectrum, intensity) might be desirable. The LED bars can easily be replaced. Make sure that the light reaches every part of the container.
If the experiment requires that the zebrafish remains in the observation container for several hours, make sure to provide aeration. An air tube can be attached to the back right corner by using a clip. Check on the computer screen that the clip does not interfere with the recording, i.e. that the zebrafish is at no time hidden from view. In Figures 2A and 2B the black clip can be seen in the top view. Ideally, aeration should be provided before or between recordings, but not during recording as to prevent noise. Also note that switching strong aeration on or off might startle the zebrafish.
If the experiment requires keeping the zebrafish in the container overnight, it is useful to cover the observation chamber with a transparent lid to prevent the zebrafish from leaping out (which is a very common occurrence). If the cover is not removed before recording (as not to disturb the zebrafish), consider the following two points: 1) make sure that the mirror images of the light sources do not obstruct the recording (e.g. by shifting the light sources or by choosing a lid that does reflect light only minimally); 2) consider that the lid might become foggy when used overnight, especially since in this case aeration has also to be provided; one way to reduce this is to choose a lid with small holes in it.
For some experiments it might be essential to determine the baseline behavior of the zebrafish before administering a drug. In this case, the drug can be added between sessions by way of a tube (similar to the aeration tube discussed above).
Although the focus here was on recording zebrafish (Danio rerio), other fish species can, of course, also be recorded with the system. It has been used to study behaviors in (juvenile) goldfish (Carassius auratus), tiger barb (Barbus tetrazona), and green swordtail (Xiphophorus helleri).
Finally it should be noted that other dimensions of the observation tanks are principally possible. However, it would require reconfiguring the system and finding a good balance between spatial and temporal resolution when using much larger observation containers. Furthermore, the shape of the container has to be cuboid. The number of zebrafish or other experimental subjects is theoretically unlimited; however tag swapping increases with number of subjects.

Zebrafish has become an important model for biological and pharmacological research1-3. Tracking of movement and spatial distribution patterns of individual zebrafish and of shoals of zebrafish is invaluable. Older studies apply manual quantification based on recorded images4. Now, 2D automatic video tracking systems are commonly used5. However, since zebrafish and many other aquatic and nonaquatic animals move in a three-dimensional space, the interest in 3D systems is growing6. The described 3D system was first published in 20077. For technical details and comparisons with other systems see8. The basic advantage of this system as compared to other systems is that it acquires two (optionally three) synchronous and independent views using a mirroring system and that is uses a two-step calibration protocol. In the first step, the 3D-geometry of the stereo system (formed by the camera and the mirrors) is calibrated. In the second step, the nonlinear errors introduced by light refraction are corrected. When studying aquatic animals, the impact of light refraction can be considerable when left uncorrected. Other so far published systems (even newer developments9) omit the two-step calibration procedure. For technical details about object identification, occlusion with multi-object tracking, jigging noise etc. the reader is referred to the original publications8,9. The system has been validated for the study of anxiety-like behavioral responses10,11, shoaling8,11,12 and, in a preliminary study, for antagonistic behavior in zebrafish13. It has also been tested for other fish species (goldfish8, swordtail, tiger barb) and for mice9.
In the following the protocols for calibration and for using the system are described and the results of an experiment testing the effects of the NMDA-receptor noncompetitive antagonist MK-801 on shoaling zebrafish are presented as a representative example to demonstrate the type of data that can be obtained with the system.
Both in the natural environment and in aquariums, zebrafish usually swims in shoals14. Shoaling preferences seems to depend on several factors, such as rearing, sex, and food availability14,15. Interestingly, the distance between individual zebrafish in a shoal becomes smaller when signals of potential danger are present, such as alarm pheromones16 or novelty11. The shoaling paradigm is therefore also being used to test anxiolytic drugs17. Dominance hierarchies18 and social learning19 are fascinating aspect of zebrafish behavior. Because of its highly developed social behavioral character, zebrafish have also become of interest for autism research20,21.
MK-801 has been reported to alter social behavior in zebrafish5,22,23, which is reminiscent of its effects on mammals24. However, MK-801 has also many other effects on other behaviors (such as swimming behavior, inhibitory avoidance and place preference25-27). Therefore it is crucial, in initial experiments to obtain as much information as possible from basic observations. This will provide the basis for designing more targeted experiments.
Survey of the recording apparatus and software
In its current configuration, the observation container (25 cm x 25 cm x 18 cm, L x W x H), holding the zebrafish, is positioned in the observation chamber (91 cm x 46 cm x 56 cm, L x W x H). The chamber also contains a camera, a mirror which is suspended at approximately &#62;45&#176; angle above the container, and LED-light bars. Figure 1 shows a simplified diagram. The camera records both the front view and the top view (mirror) to construct the 3D trajectories of the zebrafish by determining its x-, y-, and z-coordinates. For orientation purposes, the coordinate vectors are presented on the left-front corner of the observation container (position relative to camera), which corresponds with the vectors presented on the 3D views (e.g. Figure 15) generated by the Path Reconstruction Module (see further below). The sampling rate depends on the camera and the computer. In our experiments it is approximately 40 frames/sec&#160;(fps). The spatial resolution of the recorded frames is 640 x 480 pixels (this configuration was chosen as optimal balance between spatial and temporal resolution). Windows XP or higher is required.
Before the first recording, the system has to be calibrated to determine locations of the walls of the container and the water surface, to determine the scaling factor of the recorded frames and to adjust for the sizable error generated by refraction of light when passing from water to air. In principle, calibration has only to be performed during the initial installation and after parts of the setup, such as camera or mirror, are moved or replaced. Nevertheless, it is good practice to check from time to time whether the calibration is still correct and recalibrate, if necessary.
The software consists of three modules. The Recordings Module records the frames (two example frames are shown in Figure 2). The Trajectory Module reconstructs the paths for every zebrafish. The Data Processing Module calculates a range of standard kinematic and spatial parameters. For the purpose of further analysis, the data can be imported into spreadsheet or statistical software.

<table><tbody><tr><td>AABT-3D Tracking Hardware</td><td>xyZfish</td><td>AABT II</td><td>Includes camera, video card, observation compartment with mirror and lighting</td></tr><tr><td>AABT-3D Calibration equipment</td><td>xyZfish</td><td>calib</td><td>Contains calibration panel and side mirror</td></tr><tr><td>AABT-3D Observation containers</td><td>xyZfish</td><td>cubes</td><td>&amp;nbsp;</td></tr><tr><td>AABT-3D Tracking software</td><td>xyZfish</td><td>v.1.0</td><td>&amp;nbsp;</td></tr><tr><td>Computer</td><td>optional</td><td>N/A</td><td>Windows XP or higher</td></tr><tr><td>(+)-MK-801 hydrogen maleate</td><td>Sigma</td><td>M107</td><td>&amp;nbsp;</td></tr><tr><td>Air pump</td><td>Fusion</td><td>500</td><td>&amp;nbsp;</td></tr><tr><td>Air line tubing 3/16&amp;rdquo;</td><td>Lee&amp;rsquo;s Aquarium and Pet Products</td><td>14508</td><td>&amp;nbsp;</td></tr><tr><td>Medium Binder Clips</td><td>OfficeDepot</td><td>561339</td><td>To attach airline to observation container</td></tr></tbody></table>

using an automated 3d-tracking system to record individual and shoals of adult zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

Watch this Scientific Journal Video about Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish at JoVE.com

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

using-an-automated-3d-tracking-system-to-record-individual-shoals

Research

JoVE Journal

Biology

11.0K Views.  xyZfish. The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Video: Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Developmental Biology

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

Environment

Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep deprivation, and consolidated periods of behavioral immobility. The Mexican cavefish, Astyanax mexicanus (A. mexicanus), is a model for studying trait evolution in response to environmental perturbation. A. mexicanus exist as in eyed surface-dwelling forms and multiple blind cave-dwelling populations that have robust morphological and behavioral differences. Sleep loss has occurred in multiple, independently-evolved cavefish populations. This protocol describes a methodology for quantifying sleep and locomotor activity in A. mexicanus cave and surface fish. A cost-effective video monitoring system allows for behavioral imaging of individually-housed larval or adult fish for periods of a week or longer. The system can be applied to fish aged 4 days post fertilization through adulthood. The approach can also be adapted for measuring the effects of social interactions on sleep by recording multiple fish in a single arena. Following behavioral recordings, data is analyzed using automated tracking software and sleep analysis is processed using customized scripts that quantify multiple sleep variables including duration, bout length, and bout number. This system can be applied to measure sleep, circadian behavior, and locomotor activity in almost any fish species including zebrafish and sticklebacks.

This protocol details the methodology for quantifying locomotor behavior and sleep in the Mexican cavefish. Previous analyses are extended to measure these behaviors in socially-housed fish. This system can be widely applied to study sleep and activity in other fish species.

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep ...

Behavior

Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as personality. Here, we describe a behavioral protocol designed for the study of the influence of zebrafish personality on their orientation response to multiple external environmental cues, specifically water currents and magnetic fields. This protocol aims to understand whether proactive or reactive zebrafish display different rheotactic thresholds (i.e., the flow speed at which the fish start swimming upstream) when the surrounding magnetic field changes its direction. To identify zebrafish with the same personality, fish are introduced in the dark half of a tank connected with a narrow opening to a bright half. Only proactive fish explore the novel, bright environment. Reactive fish do not exit the dark half of the tank. A swimming tunnel with low flow rates is used to determine the rheotactic threshold. We describe two setups to control the magnetic field in the tunnel, in the range of the earth&#8217;s magnetic field intensity: one that controls the magnetic field along the flow direction (one dimension) and one that allows a three-axial control of the magnetic field. Fish are filmed while experiencing a stepwise increase of the flow speed in the tunnel under different magnetic fields. Data on the orientation behavior are collected through a video-tracking procedure and applied to a logistic model to allow the determination of the rheotactic threshold. We report representative results collected from shoaling zebrafish. Specifically, these demonstrate that only reactive, prudent fish show variations of the rheotactic threshold when the magnetic field varies in its direction, while proactive fish do not respond to magnetic field changes. This methodology can be applied to the study of magnetic sensitivity and rheotactic behavior of many aquatic species, both displaying solitary or shoaling swimming strategies.

We describe a behavioral protocol designed to assess how zebrafish&#8217;s personalities influence their response to water currents and weak magnetic fields. Fishes with the same personalities are separated based on their explorative behavior. Then, their rheotactic orientation behavior in a swimming tunnel with a low flow rate and under different magnetic conditions is observed.

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as ...

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 ...

Neuroscience

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Bioengineering

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

Analysis of Zebrafish Cardiac Contraction with Virtual Reality Interaction

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Author Spotlight: High-Resolution 4D Light-Sheet Imaging and Virtual Reality in Zebrafish for Single-Cell Analysis of Heart Function

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...