The study found that exposure to the organophosphate pesticide chlorpyrifos during development reduced anxiety-related behavior in zebrafish larvae. Using a novel high-throughput behavioral assay, the researchers determined that larvae treated with low doses of chlorpyrifos showed decreased thigmotaxis, or edge-seeking behavior, without and with a visual stimulus compared to controls, indicating reduced anxiety. Treatment did not affect avoidance behavior. In a separate assay, chlorpyrifos-treated larvae also showed decreased thigmotaxis, swim speed, but no change in avoidance behavior compared to controls. The results suggest that even low levels of chlorpyrifos during development can alter anxiety behaviors through effects on the serotonergic and

1. Chlorpyrifos during development reduces anxiety-related behavior in zebrafish larvae
Holly Richendrfer1 and Robbert Créton1
Department of Molecular Biology, Cell Biology and Biochemistry1, Brown University, Providence, RI, USA.
SUMMARY
METHODS CONT’D
A large percentage of the population has been
diagnosed with neurobehavioral disorders such as •Images were imported into ImageJ (1C) , an automatic macro was
anxiety, ADHD, and autism. The role that used to split color channels (1D), subtract background (1E) , and
environmental toxicants play on their development is apply a threshold to separate larvae (1F). Scale bar = 1 cm
concerning. We found that exposure to the •X,Y coordinates of larvae obtained from Image J were imported into
organophosphate pesticide (OP), chlorpyrifos, Microsoft Excel to determine if larvae were horizontally ‘up’ or ‘down’
changed anxiety related behavior in zebrafish larvae in the dish (avoidance behavior) or in the center or edge of the well
using high-throughput behavioral analysis. (thigmotaxis behavior), and swim speed (mm/min).
INTRODUCTION RESULTS
•OPs are highly ubiquitous pesticides and are used on •Larvae treated with 1µM chlorpyrifos had tail deformations and
millions of acres of farm land in the US alone every year. Figure 1 – Behavioral Five-fish assay setup were removed from behavioral analysis.
•The role that chronic low levels of OPs have on the Five Fish Assay
development of neurobehavioral disorders is still largely
•Larvae treated with 0.01 or 0.1µM chlorpyrifos showed
unknown.
decreased thigmotaxis without (Fig 2A) and with the ‘bouncing
•OPs have been implicated in the development of ADHD,
ball’ (BB) visual stimulus compared to DMSO controls (Fig 2 B).
autism and anxiety in humans, rats and mice.
•Treatment with chlorpyrifos did not affect avoidance behavior
•Zebrafish larvae are ideally suited for high-throughput
either without or with the bouncing ball (BB) at any concentration
studies of vertebrate behavior because large numbers of
compared to DMSO controls (Fig 2 C and D).
embryos can be collected daily and directly exposed to
chemicals. One Fish Assay
•A novel high-throughput assay was created in our lab for
•Larvae treated with 0.01 or 0.1µM chlorpyrifos showed
behavioral analyses of zebrafish larvae to measure
decreased thigmotaxis and decreased swim speed
thigmotaxis and avoidance behavior a (common anxiety
compared to controls (Fig 3A and 3C) while avoidance behavior
measures). Previously, our lab has verified that anxiety can Figure 2 – Five fish assay: Edge and
was the same across all groups (Fig 3B).
be detected by thigmotaxis behavior in our assay. Avoidance behavior
DISCUSSION
METHODS •Doses of chlorpyrifos used were comparable to that in the human
•Larvae were exposed to DMSO and four concentrations diet and at levels that are known to not impact AChE levels in the
of chlorpyrifos (from 0.001-1 µM) for 7 days. zebrafish.
•Larvae were held in 6-well plates and positioned on top •Even though swim speed was decreased in two groups, the larvae
of a laptop screen . Two assays were utilized to analyze still showed similar avoidance behavior comparable to controls,
thigmotaxis, avoidance behavior, and swim speed. The indicating thigmotaxis differences are existent.
five fish assay utilized a PowerPoint presentation shown • Chlorpyrifos is known to affect serotonergic and dopaminergic
to the larvae for one hour (five fish per well) (Fig 1). systems, indicating these networks may have been modified in our
•For the first half of the assay there were no visual stimuli study.
(1A), in the second half a red ‘bouncing’ ball and a red •Avoidance behaviors were not changed in any group; this behavior
stationary ball were shown to the larvae (1B). may use different neurological mechanisms compared to
•The one fish assay utilized a blank white background thigmotaxis behavior.
with no visual stimuli (one fish per well). Figure 3 – One fish assay: Edge, Avoidance
and Speed •Even low levels of chlorpyrifos exposure during development has
•Time lapse imaging was used to capture larval placement the ability to change anxiety behaviors raising concern for the use of
within the wells . this OP.