The authors have no acknowledgements.

<ol>
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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. 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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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	<tbody>
		<tr height="63">
			<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.

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

10.9K 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

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

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

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

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.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

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

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, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

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

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

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

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

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

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

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

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

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

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

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

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

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

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.