Investigating the behavioral responses to
developmental nicotine exposure in zebrafish
Amanda Slade
Mike Simonich
Tanguay Lab
September 24, 2009
Nicotine exposure during development is
still a serious problem
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1 out of 4 women smoke at
least one cigarette during
pregnancy.
Though >4000 chemicals are
detected in cigarette smoke,
nicotine is the sole chemical
driver of habitual smoking.
Cognitive and locomotor
impairments in offspring have
been attributed to maternal
nicotine exposure.
The mechanism by which
nicotine acts developmentally
is not understood.
What does nicotine do?
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Nicotine first interacts with neuronal type
acetylcholine receptors located throughout the central
nervous system.
These receptors are most abundant in the brain.
How the binding of nicotine to these receptors during
development causes learning and locomotor deficits is
not understood.
The learning and locomotor deficits appear to persist
into adulthood.
Understanding this mechanism may provide
knowledge of how to mitigate the effects of nicotine
on development.
Example of a nicotine-induced
developmental problem
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Nicotine receptors are found
on secondary motoneurons
A zebrafish line has been
made to express green
fluorescent protein in
secondary motoneurons.
Nicotine during development
causes motoneurons to grow
differently, perhaps even to
the wrong places.
This might explain locomotor
deficits in exposed animals.
Normal
neuron axon
Nicotine
exposed
Why use zebrafish?
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Zebrafish have many genes and gene
families in common with humans.
Zebrafish develop externally = easier
to study
All organs are fully formed in 120
hours.
Embryos are transparent.
>10,000 embryos can be produced
every day in the Tanguay lab
Project goal
 Develop
an automated method for
measuring embryonic behavioral
endpoints
 The
method should be applicable to embryos as
young as 24 hours
 The method should be quick and able to
accommodate many embryos
Prior knowledge: Acute nicotine exposure
induces swimming behavior
•Each experiment was video-taped
and manually examined.
•This was a very labor-intensive way
to screen a simple behavior
•Having a computer keep track of
many different aspects of movement
would be a huge step forward.
Developing an automated method
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The Tanguay Lab recently
purchased the ZebraLab to
monitor embryo movement via
digital camera.
Movement analysis by
sophisticated tracking software
My project was to learn the
software and develop the lab’s
first automated screen for
nicotine-induced movement in
embryos.
What I did with the ZebraLab
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I went beyond just bends/min and measured:
 Durations
of inactivity, slow movement, and rapid
movement.
 Total distance traveled during slow and rapid movement.
 Swim responses to stimuli
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Dark to light transition
Startle
Exposure protocol
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Used 72 hpf embryos
Loaded fish embryos into a 96 well
plate.
Removed fish water and added 100
µL of 1X embryo medium.
Let fish acclimate in the ZebraBox
for 20 min
Added 100µL of 1X embryo
medium (control)or 60 µM nicotine
(final concentration 30 µM).
Tracked larval swimming for 15
min.
Analyzed results.
Nicotine increased swimming distance
24 embryos per treatment group
N=5
Nicotine increased swimming duration
24 embryos per treatment group
N=5
Does nicotine affect a physical response
to bright light?
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The Zebrabox is equipped with a white light stimulus
function. The embryos are normally tracked under IR
light which the embryos do not see.
The plate can be white light pulsed to visually
stimulate the dark acclimated embryos.
Results
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Several different stimulus settings were tried:
 Light off for 1min; light on for 1min cycled.
 Light off for 5min; light on for 1min cycled.
 Light off for 5min; burst of three flashes cycled.
No change in the nicotine exposed animals behavior
swimming behavior relative to the control animals in
any of these scenarios.
Pitfalls of the light/dark tests
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Fish that are under 5 days old do not swim
constantly so the endpoint is not very sensitive.
The fish may have responded to the light but the
camera may not have been able to pick up on the
slight movement.
Summary
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Chemical responses like simple locomotor
behavior can now be reproducibly measured in
our lab.
Behavior can be measured automatically as part
of a routine toxicology screen.
The nicotine example:
 Distance
moved was significantly increased by brief
nicotine exposure
 Duration of movement was significantly increased
by brief nicotine exposure
 This was the first automated behavioral assessment
of nicotine effects in zebrafish embryos.
Future Directions
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Optimize testing of behavioral responses to other
stimuli:
 light/dark
transitions
 startle.
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Extend behavioral testing to routinely starting
with 24 hpf embryos (= shorter age-to-screen
time and behavior at more developmental
timepoints)
Incorporate the ZebraLab into a battery of other
tox tests.
Acknowledgments
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Dr. Robert Tanguay
The Tanguay lab
Dr. Michael Simonich
Dr. Tamara Tal
Funding support: HHMI