Locomotion and Behavioral Toxicity in Larval Zebrafish: Background, Methods, and Data - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 150

Figure 12.1 Effect of time of day on locomotor activity in larval zebrafish. Larvae (n¼59) were tested
repeatedly for 10min every hour in dark (infrared illumination). Values represent mean

SEM distance
moved in cm. Letters represent statistical results (one-way repeated-measures ANOVA followed by Fisher's
PLSD test); different letters denote statistically significant differences in activity. Locomotor activity was
highest early in the morning and then decreased to a stable low level by early afternoon.

We also determined the influence of the time of day when testing took place. This
study recorded the activity of individual larva for 10min every hour frommid-morning
until late afternoon. Figure 12.1 shows the results. Activity was highest when tested in
morning hours, and then decreased to a low and stable level in the afternoon. These
results are consistent with earlier findings (MacPhail et al., 2009). It is intriguing to note
that visual thresholds for zebrafish are also lowest in the afternoon hours (Li and
Dowling, 1998); this observation may lead to a promising future research direction.
We next determined the effect of changes in lighting on locomotion. Testing
always began in darkness (infrared light) to allow any disturbances to dissipate from
transferring the plate to the recording platform. Switching from dark to light and
back to dark had a substantial effect on activity. Activity was low in light, and then
reached a high level shortly after the larvae were returned to darkness. These results
are shown in Fig. 12.2. This pattern proved to be highly reproducible. It also seemed
paradoxical at first, since zebrafish are diurnal organisms that are most active during
daylight and quiescent at night (e.g., Cahill et al., 1998; Hurd et al., 1998). Studies
have shown, however, that changes in lighting can completely override circadian
activity rhythms in zebrafish. Figure 12.2 also highlights an important methodo-
logical feature in recording locomotion. The optical recording device can sample
activity every 30 ms, and it is the change in location from one sampling period to the
next that determined the activity of a larva. With such a high level of resolution, it is
possible that many minor changes in the location could be recorded but would not
reflect bona fide locomotion in the sense of movement of the whole organism in the