Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.
Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.
Part I: Construct the OKR apparatus
Details are shown in video demonstration.
Part II: OKR of Adult Zebrafish
If your lab has constructed an OKR apparatus, the key of adult zebrafish OKR is to hold the fish. In this part, we will show you step by step.
Our apparatus composes following parts similar to others mentioned. The circular fish chamber is 7.0 cm in outer diameter, 6.5 cm in inner diameter and 7.0 cm in high. The drum around fish chamber is 8.5 cm in out diameter, 7.5 cm in inner diameter and 7.0 cm in high. It could rotate clockwise and counter-clockwise with the speed from 20~70 rotations per min (rpm). Eleven cycles of black and white stripes are drawn on a tracing paper which adhered on the outside of the drum. The light system is composed of two circle light tubes in 21 cm diameter.
Figure 1. The model of sponge. The cliff made in this middle is to make sure that the visual field was not being sheltered.
Figure 2. The angle between eye axis and body axis.
a)When the direction of grating is nasal-to-temporal, we defined the angle at the ahead of fish body is a negative angle and the angle at the fish body is a positive angle, vice versa (for brief review also see Ref. 6). Numbers in this figure represent the sequence of pins.
b)OKR analysis example from one fish in normal group. An one-second film was transferred to a series frames and the eye angles of each frame was measured with Image-pro Plus software. Each point in this figure represents each frame we measured.
Notes:
Part III. OKR of Larval Zebrafish
1. Methylcellulose
Larval zebrafish can be immobilized in 6% methylcellulose solution while keep alive for a long time. Prepare methylcellulose solution as follows.
2. Larval Zebrafish
3. Procedure of OKR
Part IV. OMR of Adult Zebrafish
Adult zebrafish OMR could also be conducted with this apparatus, just place a column in the fish chamber for preventing fish swimming across centre (showed in Fig.3a, rotating velocity is 36 rpm, black-white cycles are 11).
Figure 3. OMR of Adult Zebrafish. (a) A white plastics column was placed at the centre of fish chamber for preventing fish swimming across it. (b) The coincidence rate of adult zebrafish following the grating. The coincidence rate was defined as the proportion of the following grating time to the total recording time. The value of 50% indicates that the fish is swimming randomly while 100% indicates the fish following grating very well. Both optic nerves were crushed with a jewelry forceps about 10 seconds. The behavior of OMR appeared as a random swimming after crush.
According to the definition of OKR, it is a phenomenon that animals move their eyes to pursue a rotating grating spontaneously. If this behavior has been conducted for a long time (e.g. above 6 hours), the eye muscles may be tired and the OKR will be attenuated.
Adult zebrafish OMR has been described completely by others7. In this video, we just show you this behavior with our apparatus. Once the optic nerves are crushed, they could not follow this grating very well.
We have performed OKR behavior test in adult, larval zebrafish and OMR in adults with our simple custom-built apparatus to evaluate the visual function of zebrafish. This OKR assay of adult zebrafish in our experiment could keep strong enough within a period of 4 hours without to harm it. This may open up OKR test in adult fish function study more efficiently.
We thank Ms. Fan Shen for her support to the zebrafish rearing. This is supported by fundings to Dr. Bing Hu (Chinese Academy of Sciences, “Bai Ren Ji Hua”, NSFC 30870833 and National Basic Science Program of China, 2009CB941302).
All animal experiments were conducted in accordance with the guidelines and regulations set forth by the University of Science and Technology of China (USTC) Animal Resources Center and University Animal Care and Use Committee.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Methylcellulose | Sigma | M6385 | Viscosity 25 cP |