Biomedical Engineering Reference
In-Depth Information
gave poor correlation both with the results in OMR and OKR assays and with the
expected effects from clinical studies (Richards, personal communication). This is
likely to be due to the compounds having effects on pigmentation via toxicity
mechanisms not related to visual function.
15.3.1 Optomotor Response Assay
The OMR is the locomotor behavior of an animal induced in response to a repetitive
pattern. This response can be elicited in zebrafish by moving horizontal stripes below
long transparent chambers in which the larvae swim (Orger et al., 2000; Krauss and
Neumeyer, 2003; Maaswinkel and Li, 2003; Roeser and Baier, 2003). The larvae
swim to maintain a constant position relative to a stripe and therefore visually normal
fish accumulate at one end of the channel. The number of larvae in the final 25%, or
“pass area,” of the channel can then be quantified and the proportion of fish in the pass
area will be reduced in groups with defective visual function. At 6-7dpf, larvae will
respond to this stimulus
90% of the time (Orger et al., 2004). The OMR technique
has been used to investigate motion perception in larval zebrafish. It was shown that
inputs to motion vision derive predominantly from L (red) and M (green) cones, but
not from short-wavelength (UV and S (blue)) cones, as is also the case in higher
vertebrates (Orger and Baier, 2005). The OMR assay has a higher throughput for
evaluating the effect of compounds on zebrafish vision than the OKR assay described
in Section 15.3.2. However, compounds that affect the locomotor ability of the
zebrafish would be false positives in the OMR assay, since hypomotility will decrease
the number of larvae scored in the pass area of the chamber. Therefore, the OMR assay
may be used as a primary screen with positive compounds being further evaluated for
defects in visual function by secondary screening in OKR and for hypomotility in a
locomotor assay. The OMR assay can also be adapted for adult zebrafish although the
throughput of such an assay is very low (Fleisch and Neuhauss, 2006). To avoid
schooling behavior, single adult zebrafish are placed inside a round testing chamber
surrounded by a rotating drum containing alternating black and white stripes and the
fish then swims in the direction of the stripes.
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15.3.1.1 OMRMethod The maximum tolerated concentration (MTC) for each
compound is first determined in larval zebrafish to identify toxic concentrations such
that testing for OMR is not compromised by general toxicity. This approach has
recently been reviewed by Hutchinson et al. (2009). The MTC is determined as the
concentration at which no lethality was observed above that seen in control (untreated)
siblings. Death is assessed by the absence of a heartbeat. In addition, the following tests
are used to assess toxicity at nonlethal concentrations: (i) locomotor activity and startle
response (gross score by eye in response to tapping the dish), (ii) swim position; loss of
dorsoventral balance, and (iii) any morphological abnormalities, the most common of
which are pericardial edema, bent body, and failure to inflate swim bladder.
Larvae are exposed to the test compound in 1% (v/v) DMSO final solvent
concentration in swimming medium in 24-well plates for 5 days (from 3 to 8dpf)
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