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 http://www.fishpain.com/fish-and-pain-brain-structures.htm Gene Sequences https://phys.org/news/2015-06-role-zebrafish-larger-scale-gene.html Cell Types & Function http://www.nature.com/onc/journal/v23/n43/fig_tab/1207943f6.html Modified from: http://10e.devbio.com/article.php?id=295 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. 2762-2772. [5] Pavlidis, M., et al. Behavioral Brain Research 225, 2011, pp. 529-537. [6] Oswald, M.E., et al. Physiological and Biochemical Zoology 85, no. 6, 2012, pp. 718-728, [7] O’Connell, L.A., and Hofmann, H.A. Science 336, 2012, pp. 1154 [8] Wong, et al. Behaviour 149, 2012, pp. 1205-1240 [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. 2002 42: 517-525.