Automated High-throughput Behavioral Analyses in Zebrafish Larvae

Abstract

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

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]. In summary, the system includes a 15 megapixel Canon EOS Rebel T1i digital camera and an Acer Aspire 5517 laptop with a 15.6 inch screen to provide visual stimuli to the larvae.…”

“…Larval behavior was examined in a ‘five-lane’ plate, with 5 larvae per lane (25 larvae per plate). The five-lane plate is made using a Nunc 1-well rectangular plate (Fisher 12-565-493), 50 ml of 0.8% agarose in egg water, and a CNC-milled plastic mold [25]. Each lane is 18 mm wide, 70 mm long and 3.5 mm deep and has 60° sloping edges to avoid shadows and blind spots along the perimeter of the swimming area.…”

“…The embryos hatch from their chorion between 2 and 3 days post-fertilization (dpf). At 5 dpf, the free-swimming larvae are approximately 4 mm long, have inflated swim bladders, and display a broad range of behaviors, which can be examined in multiwell or multilane plates [19-25]. …”

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

“…The zebrafish is an excellent model to study the developmental effects of low levels of pesticides or other toxicants in order to determine behavioral and morphological changes after exposure (Eddins et al, 2010; Levin et al, 2011; Richendrfer and Creton, 2013; Richendrfer et al, 2014; Richendrfer et al, 2012b). Zebrafish embryos develop outside of the mother and can be collected daily in very large numbers and the embryos can be treated directly in a petri dish with various toxicants, drugs, or pesticides.…”