1. Differences in behavioral responses to stress in zebrafish: exploring underlying mechanisms
Jacalyn B. Russ, Ryan Y. Wong
Department of Biology, University of Nebraska at Omaha, Omaha, NE 68182
Methods
Behavioral Assay – Novel Tank Diving Test
Used to measure vertical & horizontal movement
Stressed” ↑ time in lower portion
“Non-stressed” ↑ time in upper portion
Looking at the animals with the lowest & highest behavioral stress response
n=12 control animals (not “stressed”), n=12 proactive fish, n=12 reactive fish
Animals used to assess differences in neural activity patterns & cortisol levels
Behavioral Methodology
Fish sacrificed & stored for later molecular experiments
Molecular Methodology
In situ hybridization procedure
Behavioral
Pilot Trials
Experimental Trials
Data Analysis
Molecular
Probe Synthesis
Probe Testing
in situ hybridization
Data analysis
Hormonal
Hormone assay
Examining stress coping style differences in zebrafish
Studies have shown behavioral differences in stress coping styles, but the neural mechanisms underlying these differences are not as well defined.
Objective: Examine differences in underlying neural mechanisms between the proactive and reactive stress coping styles
Hypothesis:
a) Proactive fish will spend less time in the lower portion of the Novel Tank Diving Test and will spend more time moving around.
b) In the basolateral amygdala, hippocampus, habenula, and paraventricular nucleus of the hypothalamus neural activity will differ between alternative coping styles and whole body cortisol levels will differ between 2 coping styles
Background z
Results
Reactive fish spent significantly (p<0.001) more time in the lower portion of the NTDT than the reactive fish.
On average, proactive fish swam a greater distance (p<0.001) than reactive fish.
Animals encounter, react to, & often successfully overcome stressors
Several brain regions are involved in the stress response [1,2,3]
Paraventricular nucleus of the hypothalamus (PVN), hippocampus, habenula, basolateral amygdala
Stressor leads to physiological response (Hypothalamaic-pituitary-adrenal axis)
Release of cortisol
2 stress coping styles have been identified across taxa: Proactive & Reactive [4,5,6]
Barton, B. 2002
Measures of Movement * *
Consistency Across Time
Proactive fish did not spend significantly more time (p>0.05) in the lower portion of the NTDT during the first 6 minutes compared to the final 6 minutes of the trial.
Reactive fish spent significantly more time (p<.001) in the lower portion of the NTDT during the first 6 minutes compared to the final 6 minutes of the trial.
Proactive
Reactive
↑ exploratory behavior
↓ exploratory behavior
↓ glucocorticoid production (cortisol)
↑ glucocorticoid production (cortisol)
Rely on previous experience
Rely on environmental cues
Sex Differences
Males did not spend significantly more time (p> 0.05) in the lower portion of the NTDT than females.
Zebrafish Model
Male and female zebrafish
Becoming a more widely-used model organism for neurobiological research.[7,9]
Develop analogous human health diseases [10]
Highly similar:
Distinct, whole-brain neurotranscriptome profiles between proactive & reactive strains [11].
Stress coping styles in Danio rerio (zebrafish) [8]
Reactive & proactive lines of zebrafish
Lines showed consistent behaviors across several contexts [8]
Conclusion and Future Directions
Proactive fish spend more time in the upper portion of the NTDT and more time moving around than reactive fish.
Strain differences in behavior → should see differences in brain
Time spent in lower portion of NTDT is consistent for proactive, but not reactive fish
Reactive fish may have habituated to testing apparatus
Males did not spend more time in the lower portion of NTDT than females
Currently, the in situ hybridization process is being optimized.
Bodies will be homogenized for hormone assays
Will measure levels of whole-body cortisol a) b) c) d)
Brain areas to be analyzed using in situ hybridization
a) Habenula b) paraventricular nucleus of the hypothalamus c) hippocampus d) basolateral amygdala
Neuroanatomy
Gene Sequences
Cell Types & Function
Modified from:
Acknowledgements
I would like to thank Danny Revers and Sandra Roundtree for zebrafish husbandry. Additionally, I would like to thank the members of the Wong laboratory for feedback and overall support throughout the entire project. Finally, I would like to thank Dr. Ryan Wong and the biology department for all of their guidance and support. This project is funded by Biology Department funds to JBR and UNO UCRCA and start-up funds to RYW.
References
[1] McEwen, B. S. (2007). Physiological Reviews, 87, 873–904.
[2] Hikosaka, O. (2011). Nature, 4(164), 503–513.
[3] Gale, G. D., et al. Journal of Neuroscience, 24(15), 3810–3815.
[4] Sih, A., and Giudice, M.D., Philosophical Transactions of the Royal Society B 367, 2012, pp
[5] Pavlidis, M., et al. Behavioral Brain Research 225, 2011, pp
[6] Oswald, M.E., et al. Physiological and Biochemical Zoology 85, no. 6, 2012, pp ,
[7] O’Connell, L.A., and Hofmann, H.A. Science 336, 2012, pp. 1154
[8] Wong, et al. Behaviour 149, 2012, pp
[9] Stewart, A.M, et al. Trends in Neurosciences 37, no. 5, 2014
[10] Kalueff, A.V., et al. Trends in Pharmacological Sciences 35, no. 2, 2014
[11] Wong, et al. BMC Genomics, 2015 
Barton, B., Integrative and Comparative Biology :