1. Investigating the behavioral responses to developmental nicotine exposure in zebrafish Amanda Slade Mike Simonich Tanguay Lab September 24, 2009

2. Nicotine exposure during development is still a serious problem  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.

3. What does nicotine do?  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.

4. Example of a nicotine-induced developmental problem  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

5. Why use zebrafish?  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

6. 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

7. 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.

8. Developing an automated method  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.

9. What I did with the ZebraLab  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 Dark to light transition Startle

10. Exposure protocol 1. Used 72 hpf embryos 2. Loaded fish embryos into a 96 well plate. 3. Removed fish water and added 100 µL of 1X embryo medium. 4. Let fish acclimate in the ZebraBox for 20 min 5. Added 100µL of 1X embryo medium (control)or 60 µM nicotine (final concentration 30 µM). 6. Tracked larval swimming for 15 min. 7. Analyzed results.

11. Nicotine increased swimming distance 24 embryos per treatment group N = 5

12. Nicotine increased swimming duration 24 embryos per treatment group N = 5

13. Does nicotine affect a physical response to bright light?  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.

14. Results  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.

15. Pitfalls of the light/dark tests  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.

16. Summary  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.

17. Future Directions  Optimize testing of behavioral responses to other stimuli:  light/dark transitions  startle.  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.

18. Acknowledgments  Dr. Robert Tanguay  The Tanguay lab  Dr. Michael Simonich  Dr. Tamara Tal  Funding support: HHMI