Zebrafish have quickly emerged as a species of choice in preclinical research, holding promise to advance the field of behavioral pharmacology through high-throughput experiments. Besides biological and heuristic considerations, zebrafish also constitute a fundamental tool that fosters the replacement of mammals with less sentient experimental subjects. Notwithstanding these features, experimental paradigms to investigate emotional and cognitive domains in zebrafish are still limited. Studies on emotional memories have provided sound methodologies to investigate fear conditioning in zebrafish, but these protocols may still benefit from a reconsideration of the independent variables adopted to elicit aversion. Here, we designed a fear-conditioning paradigm in which wild-type zebrafish were familiarized over six training sessions with an empty compartment and a fear-eliciting one. The fearful stimulus was represented by three zebrafish replicas exhibiting a fully synchronized and polarized motion as they were maneuvered along 3D trajectories by a robotic platform. When allowed to freely swim between the two compartments in the absence of the robotic stimulus (test session), zebrafish displayed a marked avoidance of the stimulus-paired one. To investigate whether fear conditioning was modulated by psychoactive compounds, two groups of zebrafish were administered ethanol (0.25% and 1.00%, ethanol/water, by volume) a few minutes before the test session. We observed that ethanol administration abolished the conditioned avoidance of the stimulus-paired compartment. Ultimately, this study confirms that robotic stimuli may be used in the design of fear-conditioning paradigms, which are sensitive to pharmacological manipulations.
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Datasets and codes used in the analyses are stored at the authors’ home institution and will be provided upon request.
Brewin, C. R., Andrews, B. & Valentine, J. D. Meta-analysis of risk factors for posttraumatic stress disorder in trauma-exposed adults. J. Consult. Clin. Psychol. 68, 748–766 (2000).
Read, J., van Os, J., Morrison, A. P. & Ross, C. A. Childhood trauma, psychosis and schizophrenia: a literature review with theoretical and clinical implications. Acta Psychiatr. Scand. 112, 330–350 (2005).
Schneiderman, N., Ironson, G. & Siegel, S. D. Stress and health: psychological, behavioral, and biological determinants. Annu. Rev. Clin. Psychol. 1, 607–628 (2005).
Arnau-Soler, A. et al. A validation of the diathesis-stress model for depression in Generation Scotland. Transl. Psychiatry 9, 25 (2019).
Roy, A. et al. Family environment interacts with CRHR1 rs17689918 to predict mental health and behavioral outcomes. Prog. Neuropsychopharmacol. Biol. Psychiatry 86, 45–51 (2018).
Brett, Z. H. et al. Serotonin transporter linked polymorphic region (5-HTTLPR) genotype moderates the longitudinal impact of early caregiving on externalizing behavior. Dev. Psychopathol. 27, 7–18 (2015).
Carola, V. & Gross, C. Mouse models of the 5-HTTLPR × stress risk factor for depression. Curr. Top. Behav. Neurosci. 12, 59–72 (2012).
Bartolomucci, A. et al. Increased vulnerability to psychosocial stress in heterozygous serotonin transporter knockout mice. Dis. Model. Mech. 3, 459–470 (2010).
Gilbertson, M. W. et al. Smaller hippocampal volume predicts pathologic vulnerability to psychological trauma. Nat. Neurosci. 5, 1242–1247 (2002).
Caspi, A. et al. Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene. Science 301, 386–389 (2003).
Belsky, J. et al. Vulnerability genes or plasticity genes? Mol. Psychiatry 14, 746–754 (2009).
Bateson, P. et al. Developmental plasticity and human health. Nature 430, 419–421 (2004).
Zoratto, F. et al. Effects of maternal L-tryptophan depletion and corticosterone administration on neurobehavioral adjustments in mouse dams and their adolescent and adult daughters. Prog. Neuropsychopharmacol. Biol. Psychiatry 35, 1479–1492 (2011).
Zoratto, F., Fiore, M., Ali, S. F., Laviola, G. & Macrì, S. Neonatal tryptophan depletion and corticosterone supplementation modify emotional responses in adult male mice. Psychoneuroendocrinology 38, 24–39 (2013).
Beach, F. A. The Snark was a Boojum. Am. Psychol. 5, 115–124 (1950).
Macrì, S. & Richter, S. H. The Snark was a Boojum - reloaded. Front. Zool. 12(Suppl 1), S20 (2015).
Fontana, B. D., Mezzomo, N. J., Kalueff, A. V. & Rosemberg, D. B. The developing utility of zebrafish models of neurological and neuropsychiatric disorders: a critical review. Exp. Neurol. 299, 157–171 (2018).
Freires, I. A., Sardi, Jd. C. O., Castro, D. R. D. & Rosalen, P. L. Alternative animal and non-animal models for drug discovery and development: bonus or burden? Pharm. Res. 34, 681–686 (2017).
Gerlai, R. Fish in behavior research: unique tools with a great promise! J. Neurosci. Methods 234, 54–58 (2014).
Shams, S., Rihel, J., Ortiz, J. G. & Gerlai, R. The zebrafish as a promising tool for modeling human brain disorders: a review based upon an IBNS Symposium. Neurosci. Biobehav. Rev. 85, 176–190 (2018).
Kalueff, A. V., Echevarria, D. J. & Stewart, A. M. Gaining translational momentum: more zebrafish models for neuroscience research. Prog. Neuropsychopharmacol. Biol. Psychiatry 55, 1–6 (2014).
Walters, E. T., Carew, T. J. & Kandel, E. R. Associative learning in aplysia: evidence for conditioned fear in an invertebrate. Science 211, 504–506 (1981).
Maren, S. Neurobiology of Pavlovian fear conditioning. Annu. Rev. Neurosci. 24, 897–931 (2001).
LeDoux, J. E. Emotion circuits in the brain. Annu. Rev. Neurosci. 23, 155–184 (2000).
Blank, M., Guerim, L. D., Cordeiro, R. F. & Vianna, M. R. A one-trial inhibitory avoidance task to zebrafish: rapid acquisition of an NMDA-dependent long-term memory. Neurobiol. Learn. Mem. 92, 529–534 (2009).
Kenney, J. W., Scott, I. C., Josselyn, S. A. & Frankland, P. W. Contextual fear conditioning in zebrafish. Learn. Mem. 24, 516–523 (2017).
Maximino, C. et al. Extending the analysis of zebrafish behavioral endophenotypes for modeling psychiatric disorders: fear conditioning to conspecific alarm response. Behav. Processes 149, 35–42 (2018).
Brock, A. J., Sudwarts, A., Daggett, J., Parker, M. O. & Brennan, C. H. A fully automated computer based ‘Skinner Box’ for testing learning and memory in zebrafish. Preprint at https://www.biorxiv.org/content/10.1101/110478v1 (2017).
Fontana, B. D., Cleal, M., Clay, J. M. & Parker, M. O. Zebrafish (Danio rerio) behavioral laterality predicts increased short-term avoidance memory but not stress-reactivity responses. Anim. Cogn. 22, 1051–1061 (2019).
Fontana, B. D., Cleal, M. & Parker, M. O. Female adult zebrafish (Danio rerio) show higher levels of anxiety‐like behavior than males, but do not differ in learning and memory capacity. Eur. J. Neurosci. https://onlinelibrary.wiley.com/doi/abs/10.1111/ejn.14588 (2019).
Doving, K. B. & Lastein, S. The alarm reaction in fishes–odorants, modulations of responses, neural pathways. Ann. N. Y. Acad. Sci. 1170, 413–423 (2009).
Cianca, V., Bartolini, T., Porfiri, M. & Macrì, S. A robotics-based behavioral paradigm to measure anxiety-related responses in zebrafish. PLoS One 8, e69661 (2013).
Clement, R. J. G., Macrì, S. & Porfiri, M. Design and development of a robotic predator as a stimulus in conditioned place aversion for the study of the effect of ethanol and citalopram in zebrafish. Behav. Brain Res. 378, 112256 (2019).
Ladu, F. et al. Live predators, robots, and computer-animated images elicit differential avoidance responses in zebrafish. Zebrafish 12, 205–214 (2015).
Spinello, C., Yang, Y. P., Macrì, S. & Porfiri, M. Zebrafish adjust their behavior in response to an interactive robotic predator. Front. Robot. AI 6, 38 (2019).
Neri, D., Ruberto, T., Cord-Cruz, G. & Porfiri, M. Information theory and robotics meet to study predator-prey interactions. Chaos 27, 073111 (2017).
Romano, D., Donati, E., Benelli, G. & Stefanini, C. A review on animal-robot interaction: from bio-hybrid organisms to mixed societies. Biol. Cybern. 113, 201–225 (2019).
Katsnelson, A. Advancing artificial animals. Lab Animal 47, 201–204 (2018).
Krause, J., Winfield, A. F. T. & Deneubourg, J. L. Interactive robots in experimental biology. Trends Ecol. Evol. 26, 369–375 (2011).
Porfiri, M. Inferring causal relationships in zebrafish-robot interactions through transfer entropy: a small lure to catch a big fish. Anim. Behav. Cogn. 5, 341–367 (2018).
Miller, N. & Gerlai, R. From schooling to shoaling: patterns of collective motion in zebrafish (Danio rerio). PLoS One 7, e48865 (2012).
Hunt, P. S., Levillain, M. E., Spector, B. M. & Kostelnik, L. A. Post-training ethanol disrupts trace conditioned fear in rats: effects of timing of ethanol, dose and trace interval duration. Neurobiol. Learn. Mem. 91, 73–80 (2009).
Gould, T. J. Ethanol disrupts fear conditioning in C57BL/6 mice. J. Psychopharmacol. 17, 77–81 (2003).
Gerlai, R., Lahav, M., Guo, S. & Rosenthal, A. Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects. Pharmacol. Biochem. Behav. 67, 773–782 (2000).
Pannia, E., Tran, S., Rampersad, M. & Gerlai, R. Acute ethanol exposure induces behavioural differences in two zebrafish (Danio rerio) strains: a time course analysis. Behav. Brain Res. 259, 174–185 (2014).
Spinello, C., Macrì, S. & Porfiri, M. Acute ethanol administration affects zebrafish preference for a biologically inspired robot. Alcohol 47, 391–398 (2013).
Tran, S. & Gerlai, R. Time-course of behavioural changes induced by ethanol in zebrafish (Danio rerio). Behav. Brain Res. 252, 204–213 (2013).
Tran, S., Nowicki, M., Fulcher, N., Chatterjee, D. & Gerlai, R. Interaction between handling induced stress and anxiolytic effects of ethanol in zebrafish: a behavioral and neurochemical analysis. Behav. Brain Res. 298, 278–285 (2016).
Lonsdorf, T. B. et al. Don’t fear ‘fear conditioning’: methodological considerations for the design and analysis of studies on human fear acquisition, extinction, and return of fear. Neurosci. Biobehav. Rev. 77, 247–285 (2017).
Melia, K. R., Ryabinin, A. E., Corodimas, K. P., Wilson, M. C. & Ledoux, J. E. Hippocampal-dependent learning and experience-dependent activation of the hippocampus are preferentially disrupted by ethanol. Neuroscience 74, 313–322 (1996).
Tran, S., Facciol, A. & Gerlai, R. Alcohol-induced behavioral changes in zebrafish: the role of dopamine D2-like receptors. Psychopharmacology (Berl) 233, 2119–2128 (2016).
Maximino, C. et al. Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models. Neuropharmacology 71, 83–97 (2013).
Bartolini, T., Mwaffo, V., Butail, S. & Porfiri, M. Effect of acute ethanol administration on zebrafish tail-beat motion. Alcohol 49, 721–725 (2015).
Fontana, B. D. et al. Concomitant taurine exposure counteracts ethanol-induced changes in locomotor and anxiety-like responses in zebrafish. Psychopharmacology (Berl) 237, 735–743 (2020).
Macrì, S. et al. Three-dimensional scoring of zebrafish behavior unveils biological phenomena hidden by two-dimensional analyses. Sci. Rep. 7, 1962 (2017).
Bartolini, T. et al. Zebrafish response to 3D printed shoals of conspecifics: the effect of body size. Bioinspir. Biomim. 11, 026003 (2016).
Papaspyros, V., Bonnet, F., Collignon, B. & Mondada, F. Bidirectional interactions facilitate the integration of a robot into a shoal of zebrafish Danio rerio. PLoS One 14, e0220559 (2019).
De Lellis, P. et al. Model-based feedback control of live zebrafish behavior via interaction with a robotic replica. IEEE Trans. Robot. 36, 28–41 (2020).
Macrì, S. et al. Moderate neonatal stress decreases within-group variation in behavioral, immune and HPA responses in adult mice. PLoS One 2, e1015 (2007).
Cahill, G. M. Circadian regulation of melatonin production in cultured zebrafish pineal and retina. Brain Res. 708, 177–181 (1996).
Kim, C., Ruberto, T., Phamduy, P. & Porfiri, M. Closed-loop control of zebrafish behaviour in three dimensions using a robotic stimulus. Sci. Rep. 8, 657 (2018).
Ruberto, T., Mwaffo, V., Singh, S., Neri, D. & Porfiri, M. Zebrafish response to a robotic replica in three dimensions. R. Soc. Open Sci. 3, 160505 (2016).
Hartley, R. & Zisserman, A. Multiple View Geometry in Computer Vision. (Cambridge University Press, New York, NY, USA, 2003).
Feng, C., Xiao, Y., Willette, A., McGee, W. & Kamat, V. R. Vision guided autonomous robotic assembly and as-built scanning on unstructured construction sites. Autom. Constr. 59, 128–138 (2015).
Szeliski, R. Introduction. In Computer Vision: Algorithms and Applications. (eds Gries, D. & Schneider, F. B.) 1–25 (Springer-Verlag, London, UK, 2011).
The authors are grateful to Vrishin Rajiv Soman for his help in experiments and data collection and to Andrea Scaramuzzi for technical assistance. This work was supported by the National Institutes of Health, National Institute on Drug Abuse under grant number 1R21DA042558-01A1 and the Office of Behavioral and Social Sciences Research that co-funded the National Institute on Drug Abuse grant.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figures 1–3
Trajectory of the shoal of replicas used to control the robotic platform during the experiments
Sample tracking video of a fish from the top and front views overlaid with instantaneous speed, acceleration and angular speed. The green cross shows the position of the fish detected by the tracking software.
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Macrì, S., Karakaya, M., Spinello, C. et al. Zebrafish exhibit associative learning for an aversive robotic stimulus. Lab Anim 49, 259–264 (2020). https://doi.org/10.1038/s41684-020-0599-9
Biological Cybernetics (2021)