On the edge: Pharmacological evidence for anxiety-related behavior in zebrafish larvae

Abstract

Zebrafish larvae are ideally suited for high-throughput analyses of vertebrate behavior. The larvae can be examined in multiwell plates and display a range of behaviors during early development. Previous studies have shown that zebrafish larvae display a preference for the edge of the well and several lines of evidence suggest this edge preference (thigmotaxis) may be a measure of anxiety. In the present study, we further examined the relation between edge preference and anxiety by imaging zebrafish larvae exposed to three psychoactive drugs diazepam (Valium), fluoxetine (Prozac), and caffeine. The edge preference was first examined in a five-fish assay, with and without visual stimuli. Diazepam, a benzodiazepine that binds to GABA receptors, reduced the larval edge preference, with or without visual stimuli. In contrast, fluoxetine, a selective serotonin reuptake inhibitor, did not affect the edge preference. Caffeine increased the preference for the edge in response to visual stimuli. Similar effects were observed in a two-fish assay; diazepam-exposed larvae showed a reduced edge preference and caffeine exposed larvae showed an increased edge preference. These results suggest that the edge preference in zebrafish larvae is a measure of anxiety and further illustrate that the pharmaceuticals used in the study have different mechanisms of action. Although there are substantial differences between zebrafish and human brains, our results indicate that the signals that regulate anxiety are similar on a molecular level. We propose that high-throughput assays in zebrafish may be used to uncover genetic or environmental factors that cause anxiety disorders and may contribute to the development of novel strategies to prevent or treat such disorders.

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

“…We later confirmed that thigmotaxis behavior in this assay is a measure of anxiety-related behavior in zebrafish larvae 17 . There were significant differences in the larvae movement away from the ball and preference for the edge when compared to the blank white background.…”

“…Many trials have been performed to optimize the results of the assay, such as color of stimulus used, ideal number of larvae per lane, and length of behavioral assay. Previously, we used multi-well plates (with 6 or 12 wells) 15,17,18 . However, recently we have created the novel lane mold to create a larger swimming space for the larvae allowing us to gather a larger number of behavioral measures in a single assay 16 ( Figure 5).…”

“…A PowerPoint presentation is used as an aversive stimulus for the larvae. In the past moving red balls were shown to zebrafish larvae in 6 or 12 multi-well plates 15,17,18 . The current PowerPoint starts with a blank white background for 15 min, followed by 15 min of a moving red bar on the top half of the plate (Figure 4).…”

“…The system allows us to quickly and efficiently test subtle behavioral abnormalities after embryonic exposure to a number of pharmaceuticals and environmental toxicants [16][17][18] .…”

“…Larvae can be housed in up to four multi-well or multi-lane plates which are positioned on top of the laptop screen. We use a PowerPoint presentation as an aversive stimulus to which the larvae respond by moving away (avoidance) and swimming towards the edge (thigmotaxis) 15,17 . The images are imported into ImageJ in which an automated macro is used to split the color channels, subtract the background, and apply a threshold to identify individual larvae.…”