Understanding neurobehavioral effects of acute and chronic stress in zebrafish

Figures & data

Figure 1. Common behavioral tests to assess stress/anxiety in zebrafish (see Table 1 for examples of potential stressors).

Table 1. Selected acute stress-induced behaviors assessed in zebrafish tests (see Figure 1 for examples).

Behaviors tests	Acute stressor	Parameters	References
Aggression	Net chasing	More time near the mirror	(Giacomini et al., 2016)
Cognition	Alarm pheromone or predator exposure	Impaired spatial memory	(Gaikwad et al., 2011)
Locomotor activity	Net chasing	Longer distance traveled	(Giacomini et al., 2016)
Anxiety	Net chasing	More time in the bottom	(Giacomini et al., 2016; Mocelin et al., 2015)
	Net chasing	Reduced distance traveled	(Mocelin et al., 2015)
	Alarm pheromone	More time in the bottom	(Egan et al., 2009)
	Alarm pheromone	More freezing behavior	(Egan et al., 2009)
	Alarm pheromone	More erratic movements	(Egan et al., 2009)
	Alarm pheromone	Longer time in the dark	(Maximino, Lima, Costa, Guedes, & Herculano, 2014)
Social interaction	Net chasing	Reduced conspecific preference	(Giacomini et al., 2016)

Table 2. Comparison of behavioral and neuroendocrine effects of acute vs. chronic stress in zebrafish.

Effects	Acute	References	Chronic	References
Behavioral
Anxiety-like behavior	↑	(Giacomini et al., 2016)	↑	(Marcon et al., 2016)
Locomotor activity	↑	(Giacomini et al., 2016)	↓	(Song et al., 2018)
Aggressive behavior	↑	(Giacomini et al., 2016)	↑^a	(Rambo et al., 2017)
Memory/Cognition	↓	(Gaikwad et al., 2011)	↓	(Piato, Capiotti, et al., 2011)
Social interaction	↓	(Giacomini et al., 2016)	↓	(Piato, Capiotti, et al., 2011)
Neurochemical/neurobiological
Cortisol levels	↑	(de Abreu et al., 2014)	↑	(Marcon et al., 2016)
Serotonin levels	↓	(Shams et al., 2017)	↓	(Shams et al., 2015)
Dopamine levels	n/a		0	(Shams et al., 2015)
Brain-derived neurotrophic factor (BDNF)	↑	(Pavlidis et al., 2015)	↑	(Song et al., 2018)
Number of dendritic spines	n/a		↑	(Song et al., 2018)

n/a: data not available; 0: no effects.

^a In male, but not in female, zebrafish.

Table 3. Selected examples of pharmacological modulation of zebrafish cortisol levels in acute and chronic stress.

Model	Details	Effects on whole-body cortisol	References
Acute stress response
Alarm pheromone	15 min	Increased	(Abreu et al., 2017)
Visual contact with the predator^a	60 min	Increased	(Oliveira et al., 2013)
Exposure to blood of conspecifics	15 min	Increased	(Abreu et al., 2017)
Net chasing stress	2 min	Increased	(de Abreu et al., 2014)
Spatial restriction in a microtube	15 min	Increased	(Abreu et al., 2017)
Air exposure	1 min	Increased	(Abreu et al., 2015)
Drugs
Aripiprazole	0.556 and 556 ng/L	Blunted net chasing effect	(Barcellos et al., 2016)
Diazepam	16 µg/L	Blunted net chasing effect	(de Abreu et al., 2014)
Ethanol	0.25% and 0.5%	Blunted predator^a exposure effect	(Oliveira et al., 2013)
Fluoxetine	1, 25, and 50 µg/L	Blunted net chasing effect	(de Abreu et al., 2014)
Fluoxetine	1 µg/L	Blunted air exposure effect	(Abreu et al., 2015)
Fluoxetine	100 μg/L	Lower whole-body cortisol	(Egan et al., 2009)
Ibogaine	10 and 20 mg/L	Unaltered	(Cachat et al., 2013)
Lysergic acid diethylamide (LSD)	250 μg/L	Increased	(Grossman et al., 2010)
Mescaline	20 mg/L	Unaltered	(Kyzar et al., 2012)
Phencyclidine	3 mg/L	Increased	(Kyzar et al., 2012)
Risperidone	170 mg/L	Blunted net chasing effect	(Idalencio et al., 2015)
Chronic stress response
A battery of stressors^b	Several weeks	Increased	(Marcon et al., 2016; Song et al., 2018)
Drugs
Bromazepam	0.5 mg/L	Blunted chronic stress effect	(Marcon et al., 2016)
Fluoxetine	0.01 and 0.1 mg/L	Blunted chronic stress effect	(Marcon et al., 2016; Song et al., 2018)
Nortriptyline	0.01 mg/L	Blunted chronic stress effect	(Marcon et al., 2016)

^a Tiger Oscar fish (Astronotus ocellatus).

^b For example, net chasing, predator exposure, hunger and darkness.

Table 4. Selected open questions related to zebrafish models of stress-related disorders.

Questions

What are the exact mechanisms of individual differences in zebrafish stress responses? Do individual stress-sensitivity traits show stability across acute vs. chronic stress models in zebrafish? Are there neurobiological differences between acute short vs. acute prolonged stress in zebrafish? Are there neurobiological differences between single long-term vs. repeated chronic stress in zebrafish? How does zebrafish “personality” contribute to the expression of their stress-related phenotypes? Do specific acute stressors (e.g. alarm pheromone vs. net chasing) evoke distinct (e.g. distinguishable fear-like vs. more generalized, anxiety-like) affective phenotypes in zebrafish? Does chronic stress evoke pathological generalized anxiety-like, depression-like states or their comorbidity in zebrafish models? Surprisingly, many patterns of zebrafish responses to acute and chronic stress (Table 2) are generally similar. What are biological mechanisms for such similarity, and what molecular mechanisms may still be different in zebrafish between the two types of stress? (e.g. see (Kirsten et al., 2020) for discussion) Do acute and chronic types of stress induce resilience in the same (or different) zebrafish individuals? Can high (or low) responsivity to acute stress be a reliable predictor for zebrafish responsivity to chronic stress? Can mechanisms of resilience to acute stress share some mechanisms with resistance to chronic stress? How do acute- and chronic stress mechanisms interact with zebrafish nociception? Are there unique, acute stress- vs. chronic stress-specific mechanisms of resilience? Do zebrafish stress behaviors display overt and consistent/stable sex-specific differences? How to develop zebrafish models of stress-related disorders that would display sex differences in pathogenesis similar to those in humans? What is the role of epigenetic modulation in zebrafish acute vs. chronic stress responses? Does acute single stress induce lasting epigenetic signatures in zebrafish CNS? Does chronic stress evoke similar or distinct types of epigenetic responses? How relevant are the existing zebrafish stress-based behavioral assays to human stress disorders? How can we objectively link quantitative characteristics of zebrafish stress behavior to symptoms of human stress disorders? Are there differences in acute and/or chronic stress phenotypes presented by zebrafish in natural habitat vs. laboratory? Did zebrafish domestication affect acute and chronic stress-evoked phenotypes in similar (or distinct) ways? How can computer technologies (e.g. behavioral visualization software and artificial intelligence) lead to automatic recognition and/or extraction of zebrafish stress-like behaviors? What are shared and distinct neural circuits underlying the effects of acute vs. chronic stress in zebrafish? Can current genetic tools (e.g. CRISPR) improve our understanding of stress responses in zebrafish? What is the impact of gut microbiota on stress-related behavioral deficits in zebrafish? What is the impact of metabolic factors and food (e.g. fed vs. hungry fish) on stress-related behavioral deficits in zebrafish? Are there reliable morphological biomarkers of acute and chronic stress in zebrafish CNS? How do social experimental factors affect zebrafish stress-related phenotypes? What is the best model (e.g. group or individual testing) to assess stress-related behaviors in zebrafish? Are there stable, well-defined developmental trajectories of zebrafish stress responses? Do behavioral and molecular stress-related phenotypes change across the development and the lifespan of zebrafish? Does early (developmental) adversity/stress lead to increased or decreased stress reactivity in adulthood? Can some of experimental confounds related to zebrafish behavioral drug screening be resolved with repeated or prolonged habituation to experimental conditions prior to actual drug exposure procedure? Does acute stress in zebrafish affect their cognitive phenotypes in a manner similar (or different) from chronic stress?

Giacomini, A.C.V.V., Abreu, M.S., Giacomini, L.V., Siebel, A.M., Zimerman, F.F., Rambo, C.L., … Barcellos, L.J.G. (2016). Fluoxetine and diazepam acutely modulate stress induced-behavior. Behavioural Brain Research, 296, 301–310. doi:10.1016/j.bbr.2015.09.027

 PubMed Web of Science ®Google Scholar

Gaikwad, S., Stewart, A., Hart, P., Wong, K., Piet, V., Cachat, J., & Kalueff, A.V. (2011). Acute stress disrupts performance of zebrafish in the cued and spatial memory tests: The utility of fish models to study stress–memory interplay. Behavioural Processes, 87, 224–230. doi:10.1016/j.beproc.2011.04.004

 PubMed Web of Science ®Google Scholar

Mocelin, R., Herrmann, A.P., Marcon, M., Rambo, C.L., Rohden, A., Bevilaqua, F., … Piato, A.L. (2015). N-acetylcysteine prevents stress-induced anxiety behavior in zebrafish. Pharmacology Biochemistry and Behavior, 139, 121–126. doi:10.1016/j.pbb.2015.08.006

 PubMed Web of Science ®Google Scholar

Egan, R.J., Bergner, C.L., Hart, P.C., Cachat, J.M., Canavello, P.R., Elegante, M.F., … Kalueff, A.V. (2009). Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behavioural Brain Research, 205, 38–44. doi:10.1016/j.bbr.2009.06.022

 PubMed Web of Science ®Google Scholar

Maximino, C., Lima, M.G., Costa, C.C., Guedes, I.M.L., & Herculano, A.M. (2014). Fluoxetine and WAY 100,635 dissociate increases in scototaxis and analgesia induced by conspecific alarm substance in zebrafish (Danio rerio Hamilton 1822). Pharmacology Biochemistry and Behavior, 124, 425–433. doi:10.1016/j.pbb.2014.07.003

 PubMed Web of Science ®Google Scholar

Marcon, M., Herrmann, A.P., Mocelin, R., Rambo, C.L., Koakoski, G., Abreu, M.S., … Piato, A.L. (2016). Prevention of unpredictable chronic stress-related phenomena in zebrafish exposed to bromazepam, fluoxetine and nortriptyline. Psychopharmacology, 233, 3815–3824. doi:10.1007/s00213-016-4408-5

 PubMed Web of Science ®Google Scholar

Song, C., Liu, B.-P., Zhang, Y.-P., Peng, Z., Wang, J., Collier, A.D., … Kalueff, A.V. (2018). Modeling consequences of prolonged strong unpredictable stress in zebrafish: Complex effects on behavior and physiology. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 81, 384–394. doi:10.1016/j.pnpbp.2017.08.021

 PubMed Web of Science ®Google Scholar

Rambo, C.L., Mocelin, R., Marcon, M., Villanova, D., Koakoski, G., de Abreu, M.S., … Bonan, C.D. (2017). Gender differences in aggression and cortisol levels in zebrafish subjected to unpredictable chronic stress. Physiology & Behavior, 171, 50–54. doi:10.1016/j.physbeh.2016.12.032

 PubMed Web of Science ®Google Scholar

Piato, Â.L., Capiotti, K.M., Tamborski, A.R., Oses, J.P., Barcellos, L.J.G., Bogo, M.R., … Bonan, C.D. (2011). Unpredictable chronic stress model in zebrafish (Danio rerio): Behavioral and physiological responses. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 35, 561–567. doi:10.1016/j.pnpbp.2010.12.018

 PubMed Web of Science ®Google Scholar

de Abreu, M.S., Koakoski, G., Ferreira, D., Oliveira, T.A., Rosa, J.G.S.D., Gusso, D., … Barcellos, L.J.G. (2014). Diazepam and fluoxetine decrease the stress response in zebrafish. PLoS One, 9, e103232. doi:10.1371/journal.pone.0103232

 PubMed Web of Science ®Google Scholar

Shams, S., Seguin, D., Facciol, A., Chatterjee, D., & Gerlai, R. (2017). Effect of social isolation on anxiety-related behaviors, cortisol, and monoamines in adult zebrafish. Behavioral Neuroscience, 131, 492–504. doi:10.1037/bne0000220

 PubMed Web of Science ®Google Scholar

Shams, S., Chatterjee, D., & Gerlai, R. (2015). Chronic social isolation affects thigmotaxis and whole-brain serotonin levels in adult zebrafish. Behavioural Brain Research, 292, 283–287. doi:10.1016/j.bbr.2015.05.061

 PubMed Web of Science ®Google Scholar

Pavlidis, M., Theodoridi, A., & Tsalafouta, A. (2015). Neuroendocrine regulation of the stress response in adult zebrafish, Danio rerio. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 60, 121–131. doi:10.1016/j.pnpbp.2015.02.014

 PubMed Web of Science ®Google Scholar

Abreu, M.S., Giacomini, A., Koakoski, G., Piato, A.L.S., & Barcellos, L.J.G. (2017). Divergent effect of fluoxetine on the response to physical or chemical stressors in zebrafish. PeerJ, 5, e3330. doi:10.7717/peerj.3330

 PubMed Web of Science ®Google Scholar

Oliveira, T.A., Koakoski, G., Kreutz, L.C., Ferreira, D., Rosa, J.G.S.D., de Abreu, M.S., … Barcellos, L.J.G. (2013). Alcohol impairs predation risk response and communication in zebrafish. PLoS One, 8, e75780. doi:10.1371/journal.pone.0075780

 PubMed Web of Science ®Google Scholar

Abreu, M.S., Giacomini, A.C.V., Koakoski, G., Oliveira, T.A., Gusso, D., Baldisserotto, B., & Barcellos, L.J.G. (2015). Effects of waterborne fluoxetine on stress response and osmoregulation in zebrafish. Environmental Toxicology and Pharmacology, 40, 704–707. doi:10.1016/j.etap.2015.09.001

 PubMed Web of Science ®Google Scholar

Barcellos, H.H.D., Kalichak, F., da Rosa, J.G.S., Oliveira, T.A., Koakoski, G., Idalencio, R., … Barcellos, L.J.G. (2016). Waterborne aripiprazole blunts the stress response in zebrafish. Scientific Reports, 6, 37612. doi:10.1038/srep37612

 PubMed Web of Science ®Google Scholar

Cachat, J., Kyzar, E.J., Collins, C., Gaikwad, S., Green, J., Roth, A., … Kalueff, A.V. (2013). Unique and potent effects of acute ibogaine on zebrafish: The developing utility of novel aquatic models for hallucinogenic drug research. Behavioural Brain Research, 236, 258–269. doi:10.1016/j.bbr.2012.08.041

 PubMed Web of Science ®Google Scholar

Grossman, L., Utterback, E., Stewart, A., Gaikwad, S., Chung, K.M., Suciu, C., … Kalueff, A.V. (2010). Characterization of behavioral and endocrine effects of LSD on zebrafish. Behavioural Brain Research, 214, 277–284. doi:10.1016/j.bbr.2010.05.039

 PubMed Web of Science ®Google Scholar

Kyzar, E.J., Collins, C., Gaikwad, S., Green, J., Roth, A., Monnig, L., … Kalueff, A.V. (2012). Effects of hallucinogenic agents mescaline and phencyclidine on zebrafish behavior and physiology. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 37, 194–202. doi:10.1016/j.pnpbp.2012.01.003

 PubMed Web of Science ®Google Scholar

Idalencio, R., Kalichak, F., Rosa, J.G.S., Oliveira, T.A.D., Koakoski, G., Gusso, D., … Barcellos, L.J.G. (2015). Waterborne risperidone decreases stress response in zebrafish. PLoS One, 10, e0140800. doi:10.1371/journal.pone.0140800

 PubMed Web of Science ®Google Scholar