Poster presentation for Society for Neuroscience, 2011

1. A Novel High-Throughput Method to Detect Anxiety-Related Behaviors in Zebrafish Larvae
Holly Richendrfer 1, Sean Pelkowski1, Ruth M.Colwill 2, and Robbert Créton 1
Department of Molecular Biology, Cell Biology and Biochemistry1, Cognitive, Linguistic, and Psychological Sciences2, Brown University, Providence, RI, USA.
SUMMARY RESULTS
Thigmotaxis, a preference for the periphery, is a Diazepam
measure of anxiety in rodents. We found that a
•Larvae treated with either DMSO or diazepam show a similar
preference for the edge and anxiety are also linked in
avoidance response to the ‘bouncing ball’ visual stimulus (Fig 2
zebrafish larvae, which makes it possible to examine
A)
anxiety-related behavior in multiwell plates using
•Larvae treated with diazepam (Valium) show a reduced
high-throughput assays.
preference for the edge compared to controls, with or without
INTRODUCTION the visual stimulus(Fig 2 B)
•Zebrafish larvae are ideally suited for high-throughput Fluoxetine
studies of vertebrate behavior.
Figure 1 – Behavioral Five-fish assay setup •Larvae treated with fluoxetine (Prozac) show a reduced
•Large numbers of embryos can be collected daily and
directly exposed to chemicals preference for the bottom of the well – decreased avoidance
•A novel high-throughput assay was created in our lab for response (Fig 3 A)
behavioral analyses of zebrafish larvae •Larvae treated with either DMSO or fluoxetine show similar
•Thigmotaxis and avoidance behavior are common preference for the edge of the well (Fig 3B)
measures of anxiety-related behaviors in fish and rodents Caffeine
•Three psychoactive drugs were tested in zebrafish larvae
•Larvae treated with caffeine show a reduced preference for the
to determine their effects on anxiety behavior
bottom of the well – decreased avoidance response (Fig 4 A)
METHODS •Larvae treated with caffeine show increased preference for
Figure 2 – Avoidance and Edge - Diazepam
the edge of the well (Fig 4 B)
•7 day old larvae were treated with 100mg/L caffeine, 5 mg/L
diazepam, and 2 mg/L fluoxetine for 2 hours prior to
behavioral analysis DISCUSSION
•Larvae were held in 6-well plates (5 larvae per well) and
•Thigmotaxis behavior may be the most reliable measure of
positioned on top of a laptop screen . A PowerPoint
anxiety; it is reduced by diazepam, a common anxiolytic, and
presentation was shown to the larvae for one hour (Fig 1).
enhanced by caffeine, which is known to produce anxiogenic
•For the first half of the assay there were no visual stimuli
effects in humans and rodents.
(1A), in the second half a red ‘bouncing’ ball and a red
•The different effects of the drugs on behavior are likely due to
stationary ball were shown to the larvae (1B)
their mechanisms of action; the two behavioral assays may
•Time lapse imaging was used to capture larval placement Figure 3 – Avoidance and Edge - Fluoxetine
indicate differences between fear and anxiety
within the wells
•The signals that drive avoidance and thigmotaxis in zebrafish
•Images were imported into ImageJ (1C) , an automatic
may be similar to those in human anxiety disorders
macro was used to split color channels (1D), subtract
background (1E) , and apply a threshold to separate larvae
(1F). Scale bar = 1 cm
•X,Y coordinates of larvae obtained from Image J were Funding: This project was funded by the Eunice Kennedy Shriver
imported into Microsoft Excel to determine if larvae were National Institute of Child Health and Human Development
horizontally ‘up’ or ‘down’ in the dish (avoidance behavior) or (NICHD, R01HD060647) and the National Institute of
in the center or edge of the well (thigmotaxis behavior). Environmental Health Sciences (NIEHS, R03ES017755).
Figure 4 – Avoidance and Edge - Caffeine