Abstract
High-throughput behavioral studies using larval zebrafish often assess locomotor activity to determine the effects of experimental perturbations. However, the results reported by different groups are difficult to compare because there is not a standardized experimental paradigm or measure of locomotor activity. To address this, we investigated the effects that several factors, including the stage of larval development and the physical dimensions (depth and diameter) of the behavioral arena, have on the locomotor activity produced by larval zebrafish. We provide evidence for differences in locomotor activity between larvae at different stages and when recorded in wells of different depths, but not in wells of different diameters. We also show that the variability for most properties of locomotor activity is less for older than younger larvae, which is consistent with previous reports. Finally, we show that conflicting interpretations of activity level can occur when activity is assessed with a single measure of locomotor activity. Thus, we conclude that although a combination of factors should be considered when designing behavioral experiments, the use of older larvae in deep wells will reduce the variability of locomotor activity, and that multiple properties of locomotor activity should be measured to determine activity level.
Highlights
We examined locomotor activity produced by larvae at 4 and 7 dpf because larvae at these stages of development produce beat-and-glide swimming (Buss and Drapeau, 2001; Drapeau et al, 2002)
Previous studies have shown that patterns of locomotor activity change during development
All of the measured locomotor properties were significantly different between larvae at 4 and 7 dpf (Table 1), and were less variable for 7 dpf than for 4 dpf larvae (Figure 1C), which was consistent with a previous report (Farrell et al, 2011)
Summary
The use of larval zebrafish as a model organism in highthroughput behavioral screens is rapidly expanding due, at least in part, to the development and availability of commercial and academic analytical platforms designed to assess locomotor activity in larval zebrafish (Lessman, 2002; Anichtchik et al, 2004; Lockwood et al, 2004; Zon and Peterson, 2005; Giacomini et al, 2006; Prober et al, 2006; Burgess and Granato, 2007; Cahill, 2007; Parng et al, 2007; Winter et al, 2008; Creton, 2009; MacPhail et al, 2009; Sallinen et al, 2009a,b; Irons et al, 2010; Sylvain et al, 2010; Ali et al, 2011; Cario et al, 2011; Chen et al, 2011; Farrell et al, 2011; Padilla et al, 2011; Pelkowski et al, 2011; Cowden et al, 2012; Irons et al, 2013). Several characteristics of zebrafish embryos/larvae make them amenable to high-throughput behavioral screens. The small size of zebrafish larvae (∼4 mm in length) and large number of embryos produced from a single mating permits high-throughput testing of large numbers of animals and experimental conditions simultaneously. Zebrafish larvae are useful for chemical and pharmacological/toxicological screens, as they are permeable to small molecules (Kokel et al, 2010; Rihel et al, 2010; Peterson and Fishman, 2011; Tan and Zon, 2011). There has not been, a systematic characterization of how experimental conditions affect the locomotor activity in larval zebrafish in the context of high-throughput testing
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