Analysis of Optokinetic Response in Zebrafish by Computer-Based Eye Tracking

We quantitatively studied the ontogeny of oculomotor behavior in larval fish as a foundation for studies linking oculomotor structure and function with genetics. Horizontal optokinetic and vestibuloocular reflexes (OKR and VOR, respectively) were measured in three different species (goldfish, zebrafish, and medaka) during the first month after hatching. For all sizes of medaka, and most zebrafish, Bode plots of OKR (0.065-3.0 Hz, +/-10 degrees/s) revealed that eye velocity closely followed stimulus velocity (gain > 0.8) at low frequency but dropped sharply above 1 Hz (gain 0.8) in medaka almost from hatching; but zebrafish and goldfish exhibited progressive improvement, with only the largest equaling medaka performance. Monocular visual stimulation in zebrafish and goldfish produced gains of 0.5 versus 200 degrees /s at 0.5 Hz), first in medaka followed by larger (8.11 mm) zebrafish; but it was virtually nonexistent in goldfish. Velocity storage was not observed except for an eye velocity build-up in the largest medaka. In summary, a robust OKR was achieved shortly after hatching in all three species. In contrast, larval fish seem to be unique among vertebrates tested in their lack of significant angular VOR at stages where active movement is required for feeding and survival.

Modern computer hardware makes it possible to produce visual stimuli in ways not previously possible. Arbitrary scenes, from traditional sinusoidal gratings to naturalistic 3D scenes can now be specified on a frame-by-frame basis in realtime. A programming library called the Vision Egg that aims to make it easy to take advantage of these innovations. The Vision Egg is a free, open-source library making use of OpenGL and written in the high-level language Python with extensions in C. Careful attention has been paid to the issues of luminance and temporal calibration, and several interfacing techniques to input devices such as mice, movement tracking systems, and digital triggers are discussed. Together, these make the Vision Egg suitable for many psychophysical, electrophysiological, and behavioral experiments. This software is available for free download at visionegg.org.

To characterize the quantitative properties of the optokinetic response (OKR) in zebrafish larvae as a tool to test visual performance in genetically modified larvae. Horizontal OKR was triggered in 5-day-old zebrafish larvae by stimulation with projected computer-generated gratings of varying contrast, angular velocity, temporal and spatial frequency, and brightness. Eye movements were analyzed by a custom-made eye tracker based on image analysis. The gain of the OKR slow phase was dependent on angular velocity, spatial frequency, and contrast of a moving grating, but largely independent on brightness. Eye velocity was a logarithmically linear function of grating contrast with a slope of approximately 0.8 per log unit contrast. The OKR of the larval zebrafish is not scaled for stimulus contrast and spatial frequency. These properties make the OKR a valuable tool to quantify behavioral visual performance such as visual acuity, contrast sensitivity, and light adaptation. This behavioral paradigm will be useful for analyzing visual performance in mutant and gene-knockdown larval zebrafish.