Biomedical Engineering Reference
In-Depth Information
formed in the retina. By 5dpf, the visual system is well developed according to
electrophysiological, morphological, and behavioral criteria. However, it should be
noted that while cone receptors in the zebrafish retina develop and become fully
functional relatively early, the rod receptors do not become adult-like until 30dpf
(Bilotta et al., 2001).
15.3 METHODS FOR ASSESSING VISUAL FUNCTION
IN LARVAL ZEBRAFISH
To assess visual function in zebrafish, behavioral assays have been developed that take
advantage of inherent visual reflexes in the fish, such as the optokinetic response
(OKR) and optomotor response (OMR). These assays have been successfully
employed in a number of screens to identify mutant zebrafish with defects in the
visual system (reviewed in Neuhauss, 2003). These assays have more recently also
been applied to the evaluation of the effect of compounds on visual function
(Berghmans et al., 2008; Richards et al., 2008). The OMR and OKR methods and
validation are described in Sections 15.3.1 and 15.3.2, respectively. Larvae used for
both the OMR and OKR assays are reared in the dark since rearing in light can cause
abnormal effects on the development of the zebrafish retina (Saszik et al., 2002).
Circadian rhythms can affect visual sensitivity (Li and Dowling, 1998) that can affect
assay performance. Therefore, the OMR and OKR assays should be run at a similar
time of day on each occasion. The pH, conductivity, and dissolved oxygen of the
media are monitored during the 3-8dpf exposure of larvae to compound prior to OMR
evaluation. The OECD Guideline 212 for testing of chemicals: fish, short-term
toxicity test on embryo and sac fry stages (www.oecd.org and Chapter 4) recommends
that pH should remain within the range of
0.5 pH units in an assay. The Guideline
also recommends that dissolved oxygen should be between 60% and 100% saturation
throughout a study. There is little literature on the conductivity requirements for larval
zebrafish but adults are tolerant to conductivity ranging from 400
m
S to more than
1000
S. These conditions are monitored to eliminate the possibility that effects on
visual function could be attributable to the changes in pH, conductivity, or dissolved
oxygen rather than directly to the compound.
ERG measurement of retinal processing and histology of the larval zebrafish
retina have been applied as secondary, mechanistic studies. The methodology for
ERG is described in Section 15.3.3.
Visual background adaptation (VBA) is the aggregation and dispersal of pigment
granules in the larval zebrafish skin in response to ambient light levels and is a
camouflage mechanism. VBA is controlled in response to signals from the retina via
the hypothalamus by the pituitary secreting hormones to aggregate or disperse the
melanosomes (reviewed in Balm and Groneveld, 1998). Blind zebrafish cannot sense
the light level and therefore appear darker than control larvae and this has been used to
identify blind mutant zebrafish (Neuhauss et al., 1999). However, while VBA
provides a high-throughput screening method for compound treatments, it was found
to be unsatisfactory for screening of compound for effects on visual function, since it
m
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