Locomotion and Behavioral Toxicity in Larval Zebrafish: Background, Methods, and Data - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 149

behavior can lead to incomplete accounts and faulty interpretations of both well-
being and toxicity (Morse, 1975; MacPhail, 1990). For this reason, we detail
methods for assessing the locomotion of developing zebrafish larvae and the effects
of chemical exposures.
12.5 ANALYZING LARVAL LOCOMOTION
12.5.1 Methods
In these studies, each embryo was placed in one of the 96 wells of a microtiter plate.
Testing began at 6 days post fertilization (dpf) when the larvae were swimming yet
still feeding off their yolk sacs. Larvae were maintained in an incubator (26 C with a
14:10 h light/dark cycle) until the morning of testing, at which time they were
transferred to the (darkened) testing room (ambient temperature 26 C), and placed in
a light-tight drawer for several hours. Early transfer to the testing roomminimized any
disturbance that might be associated with handling and movement of the plate. Just
before testing, the plate was carefully placed on a platform (light box) that could
provide both infrared and visible light (from light-emitting diodes) (Noldus Infor-
mation Technology, Leesburg, VA). The light box provided infrared (800-950 nm,
with a peak at 860 nm) or visible (430-700 nm) light as measured with a wideband
spectroradiometer. A camera was placed above the platform to record simultaneously
locomotion from each of the wells. A baffle was also lowered over the platform to
prevent extraneous light from impinging on the plate and interfering with activity.
Recordings of locomotion were stored on DVDs as MPEG-2 files, and later decoded
using commercial software (EthoVision version 3.1), from which locomotion was
calculated as distance moved per unit of time. The testing equipment allowed
recording of locomotion in both visible light and infrared light, which is considered
darkness because zebrafish do not see in the infrared region of the spectrum. As
elaborated below, locomotor activity is remarkably sensitive to lighting conditions
and light-dark transitions; this observation became a central feature in the paradigms
for assessing chemical substances.
12.5.2 Variables Affecting Larval Locomotion
Preliminary experiments were conducted to determine a basic protocol for screening
chemicals. These experiments are the primary focus of this chapter. They included
determining whether well location had an effect on locomotion that might be due to
differences in optical resolution of the camera or lighting intensity at the perimeter, or
possibly due to differences in activity in larvae housed in the perimeter wells not being
“surrounded” by conspecifics. An analysis of plates containing control larvae
indicated rare instances when activity due to row or column was statistically
significant (see MacPhail et al., 2009). These rare instances were considered
biologically insignificant, but subsequent dosing studies arranged concentrations
diagonally on the plate to minimize any potential contribution of well location.