The overall goal of this procedure is to behaviorally evaluate visual system function in larva and adult zebrafish in order to quickly identify mutant larvae with visual system defects or to compare visual system properties of wild type and mutant or morph treated animals. This is accomplished by first restraining body movements of the experimental animal. Larvae are embedded in methylcellulose and adult fish are anesthetized and fitted into an immobilization device.
Next place, the body restrained animal in the Vizio tracker in the center of a drum shaped screen. Vertical black and white stripes are projected onto the inside of this screen using a digital light projector. Eye positions are then recorded and analyzed automatically by the Vizio Tracker software package, which also calculates eye velocities in real time.
The final step is to process obtained data, relate eye velocities to the properties of the visual stimulus and calculate average eye velocity and standard error for each experimental condition. Though this method can provide insight into visual system properties of larval and adult zebra fish, it can also be applied to other model organisms such as medica, kpi, and virtually any other small T species. I first had the idea for this method when we used the optic kinetic response to screen for plant larvae and quickly got interested in larvae data, not completely blind, but rather visually impaired.
Such Newton larvae potentially affect aspects of visual processing. The system includes an immobilizing device for small fish positioned under a high quality video camera, equipped with a zoom lens. The fish container is surrounded by a drum screen onto which computer generated stimulus patterns are projected.
Eye movements are recorded using the camera and automatically analyzed by the Vizio Tracker software package in real time. In order to prevent body movement during recording, embed fish larvae at five days post fertilization in 3%Methylcellulose that has been warmed to 28 degrees Celsius. Pour the warm methyl cellulose into a 35 millimeter cell culture dish and then transfer the larvae using a serum pipette with a large hook.
Mix the larvae into the methyl cellulose, then use a small needle to remove air bubbles and to position the larva up to eight larvae can be prepared for experiments simultaneously. Once the larvae are immobilized, place the cell culture dish into a rack for eight dishes. Then begin projecting the stimulus stripes onto the surrounding drum to immobilize adult fish.
First, briefly anesthetize them with 300 milligrams per liter MS 2 22 in a separate tank. Once anesthetized fit the fish into the mobilization device by gently clamping the body of the fish between two pieces of sponge Properly. Restraining body movements of adult fish is the most difficult step of this procedure.
To facilitate insertion into the immobilization device, stabilize the sponge by two plastic calf pipes. Once the fish is immobilized, place the device into the Vizio tracker and allow the fish to recover for one to two minutes prior to recording. To provide a stimulus for eye movement project, a software generated stimulus pattern consisting of vertical black and white sine wave gradings onto the drum screen.
Using the digital light projector contained within the Vizio tracker, illuminate the fish from below with infrared light, and then use the software package to record the eye movements in response to the stimulus pattern. Larval bumper mutants have reduced lens size and ectopic location of the lens, and these morphological alterations are reflected by a significant reduction of contrast sensitivity when compared to wild type. As shown here, bumper mutants increasingly fail to adjust eye velocity as the stimulus contrast decreases.
Additionally, when the stimulus spatial frequency is increased by decreasing the width of the stimulus, stripe bumper mutants also demonstrate reduced visual acuity compared to wild type siblings. Adult wild type zebrafish show a marked reduction in contrast sensitivity when maintained in increasing alcohol concentrations for 30 minutes before recording. A similar dose dependent reduction of overall eye velocity is seen over overall wide range of spatial frequencies when the fish were treated with increasing alcohol concentrations.
Alcohol treatment also dose independently reduces ocular-motor performance at more demanding tasks as shown here in response to increased stimulus speeds After its development. This technique paved the way for researchers in the field of vision research to explore development and function of the visual system in Sbra fish, medical goldfish, and other model organisms using both genetic and pharmacological models.
