High-Throughput Analysis of Behavior in Zebrafish Larvae: Effects of Feeding

Abstract

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. Identification of these factors is facilitated by high-throughput analyses of behavior in zebrafish larvae, which can be imaged in multiwell or multilane plates. However, the nutritional needs of zebrafish larvae during the behavioral experiments are not fully understood. Zebrafish larvae begin feeding between 4 and 5 days postfertilization (dpf), but can live solely on nutrients derived from the yolk until at least 7 dpf. To examine whether feeding affects behavior, we measured a broad range of behaviors with and without feeding at 5, 6, and 7 dpf. We found that feeding did not have a significant effect on behavior in 5-day-old larvae. In contrast, fed 6- and 7-day-old larvae displayed increased avoidance responses to visual stimuli, increased swim speeds, and decreased resting in comparison to unfed larvae. In addition, the fed 7-day-old larvae displayed a decrease in thigmotaxis and a decrease in the distance between larvae in the presence of visual stimuli. Thus, feeding affects a range of behaviors in 6- and 7-day-old larvae. We conclude that 5-day-old larvae are well-suited for high-throughput analyses of behavior, since effects of feeding can be avoided at this time. For high-throughput analyses of behavior in older larvae, standard feeding protocols need to be developed.

Motivated state control in larval zebrafish: behavioral paradigms and anatomical substrates

“…Starvation increases the responsiveness of several fish species to prey, but actually suppresses escape from predatory threats, possibly reflecting elevated risk-tolerance during food-search behavior (Ware, 1972; Croy and Hughes, 1991; Munk, 1995). Intriguingly, similar results have already been obtained in zebrafish larvae: unfed larvae show reduced avoidance of a simulated visual threat (Clift et al, 2014). Experiments in zebrafish confirm that changes in sensory responsiveness can be highly modality specific.…”

Effects of embryonic cyclosporine exposures on brain development and behavior

“…During the first 15 minutes, behaviors are analyzed without visual stimuli. During the subsequent 15 minutes, behaviors are analyzed in the presence of an aversive visual stimulus, a red moving bar, which the larvae avoid [21, 25]. We found that day 3 cyclosporine exposures affected the avoidance response (Fig 7A), swim speed (Fig 7B), resting (Fig 7C), the average distance between larvae (Fig 7D), the percentage of time that larvae are close together (Fig 7E), and the percentage of time the larvae spend on the edge of the swimming area (Fig 7F).…”

“…The 5 dpf larvae receive nutrients from their yolk sac and effects of feeding can be avoided at this time [21]. The larvae were imaged with a custom-built imaging system, as described previously [24-25].…”

“…We developed an ImageJ macro (version 25k) that automatically separates the color channels, subtracts the background, applies a threshold, identifies larvae based on particle size, and repeats these steps for subsequent images in a series. This macro can be downloaded from Clift et al 2014 [21]. The macro generates a long list of X,Y coordinates indicating the location and orientation of the larvae over time.…”

Chlorpyrifos and malathion have opposite effects on behaviors and brain size that are not correlated to changes in AChE activity

“…Larvae were exposed during different days of development and then analyzed for behavior at 5 days post fertilization (dpf), a timepoint chosen because larval activity does not change at this age with external feeding (Clift et al, 2014). Concurrent time points were used in order to analyze AChE activity using the Ellman assay (Ellman et al, 1961).…”