1. Introduction Zebrafish (Danio rerio) have become an increasingly popular animal model for the study of anxiety. A variety of studies have already begun to examine the effects of various drugs, such as alcohol, on anxiety related behavior in zebrafish (e.g. Wong et al., 2010; Gerlai et al., 2007; Egan et al., 2009) In order to interpret the effects of alcohol and other drugs on the zebrafish brain, however, we must first better understand the behaviors being measured. The behaviors that have been used to identify fear/anxiety in zebrafish have been dark/light preference (e.g. Blaser et al., 2009; Brielmaier, et al., 2007), a diving response (e.g. Sackerman, et al., 2010), thigmotaxis (e.g. Maximino et al., 2010) and freezing (e.g. Blaser et al., 2009; Egan et al., 2009). Here we focus on the effects of three doses of ethanol on a series of potential behavioral measures of anxiety. The behaviors that were measured included freezing (or immobility), locomotor activity (path length), latency of first movement, distance from the walls, and dark/light preference. We examined behavior both in the presence of ethanol, and immediately following the removal of ethanol. The question was (1) whether all of the measured behaviors seem to cluster around a single construct (anxiety), and (2) whether alcohol appears to have an anxiolytic, or an anxiogenic, effect on the behaviors. Procedure: ♦ Experiment 1: Subjects were pre-exposed to ethanol or water for 30 minutes, and then were given a preference test (black/white)‏ for 30 minutes. Group 1 (ETOH Present): Half of the fish received pre-exposure to 0.0%, 0.25%, 0.5%, or 1.0% ethanol, and were tested in the same dose. Group 2 (ETOH Removed): Half of the fish received pre-exposure to 0.25%, 0.5%, or 1.0% ethanol, and were tested in 0.0% ethanol. ♦ Experiment 2: Subjects were pre-exposed to ethanol or water for 30 minutes. Doses used were 0.0%, 0.25%, 0.5% or 1.0%. They were then given four 30-minute tests: (1) Black/White preference pre-test, (2) confined to black, (3) confined to white, (4) Black/White preference post-test. The pre-exposure dose of ethanol was present throughout testing. ♦ All data was recorded using a video camera located above the tank. The video camera recorded the subject’s behavior: location, latency to move, proportion freezing, distance moved, and distance from the walls. ♦Analysis of Variance (ANOVA) was used to compare the different treatments. Behaviours were also correlated to seek patterns of occurrence. Results Methods Subjects: ♦ Experiment 1: 77 subjects, 9-14 fish per dose group ♦ Experiment 2: 42 subjects, 8-12 fish per dose group ♦ Mixed gender, heterogenous phenotype (wild-type) ♦ Housed in an Aquaneering table-top housing, with a recirculating filtration system using mechanical, biological, and chemical filtration (in groups of about 20 per 10L tank) Apparatus: ♦ 2L black and white rectangular tanks ♦ Preference tanks were divided into two chambers using a clear acrylic divider, with 2” x 2” door, between the black and white sides ♦ See Figure 1 Methods Subjects: ♦ Experiment 1: 77 subjects, 9-14 fish per dose group ♦ Experiment 2: 42 subjects, 8-12 fish per dose group ♦ Mixed gender, heterogenous phenotype (wild-type) ♦ Housed in an Aquaneering table-top housing, with a recirculating filtration system using mechanical, biological, and chemical filtration (in groups of about 20 per 10L tank) Apparatus: ♦ 2L black and white rectangular tanks ♦ Preference tanks were divided into two chambers using a clear acrylic divider, with 2” x 2” door, between the black and white sides ♦ See Figure 1 Conclusion : Ethanol affects two separate pathways Locomotor Behaviors: Latency to Move, Freezing, Path Length Ethanol activates locomotor system: quicker to move, less freezing, longer distance Ethanol attenuates repeat-testing effects on locomotor behavior Conventional interpretation would be Anxiolytic Spatial Behaviors: Thigmotaxis, Black/White preference Increases avoidance of white, marginal reduction of thigmotaxis Mixed interpretation: more like Anxiogenic Following removal from ethanol, ethanol-like effects remain for at least 30 minutes A brief ‘wash-out’ period before further testing is insufficient to bring the system to baseline Conclusion : Ethanol affects two separate pathways Locomotor Behaviors: Latency to Move, Freezing, Path Length Ethanol activates locomotor system: quicker to move, less freezing, longer distance Ethanol attenuates repeat-testing effects on locomotor behavior Conventional interpretation would be Anxiolytic Spatial Behaviors: Thigmotaxis, Black/White preference Increases avoidance of white, marginal reduction of thigmotaxis Mixed interpretation: more like Anxiogenic Following removal from ethanol, ethanol-like effects remain for at least 30 minutes A brief ‘wash-out’ period before further testing is insufficient to bring the system to baseline References: Blaser RE, et al. Parametric analyses of anxiety in zebrafish scototaxis. Behav Brain Res (2010), Brielmaier J.M, et al. Nicotine place preference in a biased conditioned place preference design. ScienceDirect (2007), Egan RJ, et al. Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behav Brain Res (2009), Maximo C, et al. Parametric analyses of anxiety in zebrafish. Behav Brain Res (2009), Figure 3a Ethanol present: latency to move decreases as the dose increases Removal of ethanol: latency decreases as the doses increase (main effect of dose in both) Figure 3b Latency to move decreases as dose increases in the post-test (main effect of dose) Figure 4a Ethanol present: locomotor activity increased with dose (main effect) Removal of ethanol: non-significant increase in activity Figure 4b Locomotor activity increased with dose (main effect of dose). Figure 5a Ethanol present: reduction in freezing with dose(main effect of dose). Removal of ethanol: non-significant reduction in freezing Figure 5b Freezing decreases with larger doses of ethanol. Freezing increases with multiple testing. Figure 6a No significant effects Figure 6b No significant effects of dose Further from walls in post-test (main effect of test) Effects of Ethanol on Anxiety-related Behaviors in Zebrafish (Danio rerio) Yliana M Penalosa & Rachel Blaser Department of Psychological Sciences, University of San Diego Figure 1: Diagram of the black/white preference tank used in the experiment Figure 2b Time in white decreased with higher doses (significant main effect of dose). Figure 2a Ethanol present: no effect of dose Removal of ethanol: Time in white is reduced with higher doses (significant main effect of dose) Possible Interpretations: Ethanol exposure produces fear/anxiety (as measured in the black/white test), and additionally activates the locomotor system Ethanol exposure reduces fear/anxiety (as measured by freezing/diving), and additionally affects the (poorly-understood) mechanism underlying scototaxis behavior Further investigation is necessary to determine whether locomotor behaviors, or spatial behaviors, are valid measures of anxiety in zebrafish. Additionally, it is possible that both represent alternate pathways linked to fear (i.e. hiding vs. escape behavior); if so, further characterization of how the two interact is necessary. ETOH PresentETOH RemovalMultiple Testing Proportion White Reduced Prop. W Thigmotaxis Reduced Thigmotaxis Latency to Move Reduced Latency Increased Latency Locomotor Activity Increased Activity Reduced Activity Freezing Reduced Freezing Increased Freezing Table 2: An overview of the results is outlined here. Red cells are effects that might be interpreted as anxiogenic, while green cells are effects that might be interpreted as anxiolytic. Statistically significant effects are indicated by bold font. ThigmotaxisLatency to Move Distance Moved Freezing Proportion White.25-.17-.06-.01 Thigmotaxis -.07-.02.04 Latency to Move.30.41 Distance Moved -.78 Table 1: Correlations between behaviors are presented here. It appears that Freezing, Latency to move, and mean path length are closely correlated. Proportion in White is largely independent of these locomotor variables. Statistically significant correlations are indicated by bold font.