Description

The Zebrafish Rotation test apparatus is a circular container with transparent walls surrounded by a rotating acrylic drum. This drum can be tagged with cues for zebrafish retinal degeneration experiments. Typically, a black segment is marked on the acrylic. A central post is placed to prevent the zebrafish from swimming across the midline of the innter chamber.

Optional add ons  include:

• Backlight underneath the apparatus
• Multi colored outer chamber. For custom pattern requests let us know.

Frequently Asked Questions

• Can the Rotation speed be changed?
  • Yes, the rotation is controlled with the Conductor Software (Free with your purchase).
• Is the zebrafish behavior logged?
  • The zebrafish behavior is not logged, but the Conductor Software integrates with Noldus Ethovision, allowing for video tracking dictating the behavior of the rotation chamber. Amazing!
• Can the main rotating chamber be changed in size?
  • Yes the rotation chamber can be changed within 25% of  the offered size. If you need another size please let us know and we will provide you with a reasonably priced quote.
• How to patterns change on the rotating drum?
  • Patterns are attached with paper and clips. If you need a more robust methodology we can provide acrylic circular inserts. However, only certain sizes are available. Please inquire for more details

Documentation

Introduction

Zebrafish rotation test or adult escape response test is the only behavioral assay available for the screening of adult mutant zebrafish having visual defects. This task is necessary for the quantitative analysis of the visual sensitivity of adult zebrafish. The visual sensitivity is measured in terms of the absolute visual threshold of the cones and rods.

The response is elicited when the fish is susceptible to a potential predator, simulated by a black stripe in the task.

The response is manifested by avoidance behavior, and the subject continues to move in the opposite direction of the black stripe. The zebrafish also avoids dark spots on a white background. (Ninkovic and Bally-Cuif, 2006)

In this model, the fish is introduced into a clear stationary container surrounded by a moving drum covered with white paper. The stationary container has an opaque post in the center. The outer drum is provided with a black stripe which mimics a threatening object for the zebrafish.

With the rotating outer drum, the fish continue to move to the opposite side of the opaque post, hidden from the stripe.

This behavior depends on the fish’s ability to see and can be used to evaluate visual performance. (Li and Dowling, 1997).

By fluctuating light intensity with a white light source, the progression of dark adaptation after light adaptation and absolute rod and cone threshold levels can be measured reliably.

Escape response paradigm was originally developed by Lei Li and John E. Dowling in 1997. They utilized the apparatus to screen a dominant mutation, night blindness a (nba), that triggers a slow retinal degeneration in zebrafish. (Hans Maaswinkel et.al, 2005)

Apparatus and Equipment

The apparatus consists of a transparent stationary circular container approx. 10 cm in diameter.

This stationary container is surrounded by a rotating acrylic drum wrapped with white paper. The drum is also provided with a black stripe approx. 5×5 cm that mimics as a threatening object.

The inner container has a central opaque post approx. 3cm in diameter that prevents the fish from swimming across the midline of container.

From above, the drum is illuminated with the help of a white light source and the intensity is dependent on the nature of the experiment.

The drum is rotated at 10 rpm with the aid of a belt attached to a motor. The subject’s activity can be tracked with the help of a video tracker such as Noldus Ethovision XT.      

Training Protocol

Evaluation of the visual threshold 

Before the experiment, adult zebrafish are kept in transparent containers separately. Initially, the subjects are light adapted by exposure to a bright light source having an intensity approx. 3.25×10³ μW/cm² for about 20 minutes.

After light adaptation, the subjects are completely deprived of light (dark adaptation) for about 2 minutes. Then the subjects under study are introduced to the apparatus to determine the threshold light intensity necessary to induce escape response.

Initially, the light intensity is set at log I =-3.0 which can be increased by neutral density filters by 0.5 log units. These filters are used to fluctuate the light intensity of the drum. (Li and Dowling, 1997)

If the fish failed to response to the default intensity, the light intensity is increased by half log unit, or until the fish displayed escape response. The minimum light intensity that induced escape response is documented as the absolute threshold.

Evaluation of mutant screening

For mutant screening, the light intensity is set at log I= -5.0, approximately 1 log unit above the absolute rod threshold of wild-type zebrafish. Subjects that failed to exhibit the escape response are alienated for rescreening on succeeding days. (Li and Dowling, 1997)

To assess mutant individuals in next generations, subjects are chemically mutagenized with ENU and then crossed with wild-type fish to get the F1 generation.

From F1 generation, those individuals with night blindness are crossed with wild-type fish to get the F2 generative. These two successive progenies are examined to assess the visual thresholds both for rods and cones with the help of the escape response paradigm.

Modifications

Besides Lei and Dowling, Hans Maaswinkel et.al determined three dominant night blindness mutations namely night blindness e (nbe), night blindness f (nbf), night blindness g (nfg) in 2005. (Hans Maaswinkel et.al, 2005)

Hans Maaswinkel et al. employed similar apparatus with slight modifications in dimensions and the light intensity.

The initial intensity was set at log=-7.0 and was incrementally increased by half log units until the fish depicted escape response at least five out of ten times.

Sample Data

The sample data is depicted by comparing the absolute thresholds of the control and mutant subjects. It is evident from the graph that both the absolute threshold of cones and rods in mutant subjects are raised significantly.

Strengths & Limitations

Strengths

The adult escape response model has been in use for a very long time, and there is no other apparatus or test to isolate adult mutant zebrafish on the basis of visual defects.

The method also established the positive correlation of the circadian rhythm with visual sensitivity.

The method is not just restricted to nba mutant strain. However, it has identified various mutant strains with progressive retinal degeneration. (Maaswinkel et al. 2003, 2005)

These mutations have proved to be significant regarding human model. This is because a considerable number of human outer retinal dystrophies are progressive and affect elderly patients.

Limitations

The adult escape response task is time-consuming, and skilled personnel is required to conduct the experiment.

Summary and Key Points

• Zebra rotation test is widely used to separate adult mutant zebrafish on the basis of visual sensitivity and defects.
• The apparatus consists of two containers, the inner container is transparent, whereas the outer drum is wrapped with white paper having a black segment.
• With the help of this task, the visual threshold for rods and cones is measured, and the adult mutant screening is accomplished.
• Hans winksel et.al made slight modifications to the dimensions of the apparatus and also made slight changes to the light intensity.

References

Lei Li, Dowling JE. A dominant form of inherited retinal degeneration caused by a non-photoreceptor cellspecific mutation. Proc Natl Acad Sci USA 1997; 94: 11645–11650.

Maaswinkel H, Riesbeck LE, Riley ME, Carr AL, Mullin JP, Nakamoto AT, Li L. Behavioral screening for nightblindness mutants in zebrafish reveals three new loci that cause dominant photoreceptor cell degeneration. Mech Ageing Dev. 2005; 126:1079–1089.

Fleisch V. C., Neuhauss S. C. F. Visual behavior in zebrafish. Zebrafish. 2006; 3(2):191–201. doi: 10.1089/zeb.2006.3.191.

Ninkovic J, Bally-Cuif L. The zebrafish as a model system for assessing the reinforcing properties of drugs of abuse. Methods. 2006; 39(3):262–274. doi: 10.1016/j.ymeth.2005.12.007.