Assessment of Effects on Visual Function in Larval Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 191

within the larvae using liquid chromatography-mass spectrometry would allow for
the detection of false negatives arising from lack of compound penetration. This
would also provide the effective concentrations in the larvae allowing a comparison
with the effects of compounds in other model systems and with plasma concentrations
in humans. In addition, a comparison of four standard compounds in the OMR assay in
WIK and TL zebrafish wild-type strains revealed no difference in sensitivity between
the two strains.
In a second study, the OMR assay correctly predicted the effect on visual function
in mammalian or clinical studies of seven compounds of the nine tested (Berghmans
et al., 2008). In agreement with previously observed mammalian preclinical or
clinical effects, chloroquine, chlorpromazine, diazepam, nicotine, ouabain, and
phenytoin showed inhibition of OMR. Atropine and lithiumwere the two compounds
tested that did not affect zebrafish larval OMR but are known to affect vision in
humans by affecting binocular vision or accommodation. However, zebrafish have no
binocular vision and a spherical lens with an accommodation process unlike that of
man (Vihtelic et al., 1999) and this may explain the false negative data for these
compounds. In addition, lithium is known to cause nystagmus and decreased
accommodation only after prolonged treatment in the clinic, and a 5-day exposure
in larval zebrafish may not be long enough to replicate this effect.
15.3.2 Optokinetic Response Assay
The optokinetic response, which maintains optimal visual acuity and measures the
ability of larval zebrafish to track movements in their environment, is observed as
early as 3dpf and is present in 98% of larvae at 5dpf (Brockerhoff et al., 1995, 1997;
Neuhauss et al., 1999). The OKR assay is carried out with larvae immobilized
in methylcellulose inside a drum on which black and white stripes are rotated,
which elicits the optokinetic nystagmus (Brockerhoff et al., 1995, 1997; Easter
and Nicola, 1996; Roeser and Baier, 2003; Brockerhoff, 2006; Huang and
Neuhauss, 2008). A schematic diagram of the OKR apparatus is shown in Fig. 15.4.
Nystagmus is a stereotyped behavior in which a series of smooth ocular pursuits
track the movement of the stripe, followed by a rapid saccade as the eyes flick onto
the next stripe as the first leaves the visual field. Eye movements are instantly
reversed when the direction of the rotating stripes is reversed. The movement of the
large, pigmented zebrafish eye can be easily followed under a microscope and the
number of saccades counted. Since larval zebrafish absorb oxygen through the skin,
they can be maintained in the methylcellulose for several hours without adverse
effects. Larvae with defective visual function are expected to show a reduced number
of saccades compared to control (untreated) larvae. Changing the stripe width, angular
velocity, and contrast also allows quantification of visual acuity, contrast sensitivity,
and light adaptation in zebrafish larvae. A study by Rinner et al. (Rinner et al., 2005)
revealed that optokinetic response in zebrafish larvae, as in cats and humans, is a function
of angular velocity rather than temporal frequency. The OKR assay can also be adapted
to test for color vision as demonstrated by the isolation of a red light-insensitive mutant