Name: using an automated 3d-tracking system to record individual and shoals of adult zebrafish
Uploaded: 2013-12-05T16:00:00
Duration: 14 min 3 s
Description: Watch this Scientific Journal Video about Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish at JoVE.com

The authors have no acknowledgements.

<ol>
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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. 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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.

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			<td height="63" style="height:63px;width:176px;">AABT-3D Tracking Hardware</td>
			<td style="width:195px;">xyZfish</td>
			<td style="width:119px;">AABT II</td>
			<td style="width:197px;">Includes camera, video card, observation compartment with mirror and lighting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">AABT-3D Calibration Equipment</td>
			<td style="width:195px;">xyZfish</td>
			<td style="width:119px;">calib</td>
			<td style="width:197px;">Contains calibration panel and side mirror</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">AABT-3D Observation Containers</td>
			<td style="width:195px;">xyZfish</td>
			<td style="width:119px;">cubes</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">AABT-3D Tracking Software</td>
			<td style="width:195px;">xyZfish</td>
			<td style="width:119px;">v.1.0</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Computer</td>
			<td style="width:195px;">optional</td>
			<td style="width:119px;">N/A</td>
			<td style="width:197px;">Windows XP or higher</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">(+)-MK-801 hydrogen maleate</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">M107</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Air pump</td>
			<td style="width:195px;">Fusion</td>
			<td style="width:119px;">500</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Air line tubing 3/16 in</td>
			<td style="width:195px;">Lee&#8217;s Aquarium and Pet Products</td>
			<td style="width:119px;">14508</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:176px;">Medium Binder Clips</td>
			<td style="width:195px;">OfficeDepot</td>
			<td style="width:119px;">561339</td>
			<td style="width:197px;">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.

Использование системы 3D-слежения Автоматизированная для записи отдельных и косяки взрослых данио рерио

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 ...

Research

JoVE Journal

Biology

Retro-orbital Injection in Adult Zebrafish

Ретро-орбитальный инъекций у взрослых данио рерио

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Dissection of Organs from the Adult Zebrafish

Препарирование органов у взрослых данио рерио

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Development of automated imaging and analysis for zebrafish chemical screens.

Разработка автоматизированной обработки изображений и анализа для экранов данио химических веществ.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Intraperitoneal Injection into Adult Zebrafish

Внутрибрюшинного введения в взрослых данио рерио

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Dissection of the Adult Zebrafish Kidney

Препарирование взрослых почек данио рерио

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

В естественных условиях электропорации Morpholinos в Retina взрослых данио рерио

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

В естественных условиях электропорации Morpholinos в Восстанавливающий взрослых данио рерио хвостового плавника

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Выделение и культура Индивидуальные мышечных волокон и их сателлитные клетки от взрослых скелетных мышц

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Gavaging Adult Zebrafish

Gavaging взрослых данио рерио

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...