Visual learning and its underlying neural substrate in two species of teleost fish (Zebrafish and Ambon damselfish) Emmanuel Marquez-Legorreta Bachelor in Psychology Master in Neuroscience A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2019 The Faculty of Medicine Abstract The visual sense is one of the main sources of information for many animals. In particular, many species of fish rely on vision for survival. Whether a fish needs to distinguish between possible predators, sources of food, a possible mate or competitors for their territory, learning to discriminate between visual stimuli is fundamental part of their life. Recently, studies have shown that multiple species of fish are able to solve visual discrimination tasks that were thought to be too complex for these organisms (Brown et al., 2011). Furthermore, neuroanatomical studies have also found that although the layout of the central nervous system of teleost fish is different, multiple structures seem to have homologues in mammalian brains (Mueller et al., 2011). Together, these findings have reinforced the role of fish as a model system to study the neuronal substrates of simple and complex behaviours (Gerlai, 2014). A particular case is the one of zebrafish, whose development as a model for neuroscience has been exceptionally rapid (Stewart et al., 2010; Blaser and Vira, 2014; Kalueff et al., 2014; Stewart et al., 2014a; d'Amora and Giordani, 2018). In the last decade, the combination of genetic and optical technology has allowed the neuronal activity imaging of the whole brain of zebrafish larvae (Ahrens et al., 2013a; Wolf et al., 2015; Vanwalleghem et al., 2018). These powerful technologies are presenting us for the first time with the opportunity to see and analyse the processing of visual information in the whole brain as fish learn. To accomplish the goal of analysing visual learning in the larval zebrafish’s whole brain, I chose to use a habituation paradigm. Habituation is a simple form of learning defined as a reduction of an innate response to a frequently repeated stimulus. Zebrafish larvae show habituation to visual and auditory stimuli (Best et al., 2008; Roberts et al., 2011; Wolman et al., 2011; Randlett et al., 2019), and provide an appealing platform from which to study habituation’s circuit-level mechanisms. When presented with a looming stimulus that resembles an approaching predator, larvae respond with a rapid escape behaviour. I first showed that zebrafish larvae are capable of habituation to repetitive looms, decreasing the probability of escape responses. Also, changing features of the stimulus, like speed and inter stimulus interval, modulated the habituating responses. Next, I used a selective plane illumination microscopy (SPIM) microscope and calcium imaging to visualize neuronal activity and localize the regions associated in this learning behaviour. The imaging experiments showed that different populations of neurons display a range of habituating responses across the brain, varying in their habituation rates. I then used graph theory to model the network connectivity changes during habituation, and found that some parts of the network disengage early while a smaller fragment I sustains its connectivity. A third fraction of the network, whose elements are mostly located in the optic tectum, disconnect gradually and seem to be involved in the reengagement of the network when recovery occurs. Finally, I performed habituation experiments with fmr1 mutant zebrafish larvae, a fragile X syndrome (FXS) model. The results of both behavioural experiments and network analysis of the neuronal activity suggest that fmr1 mutants habituate more slowly and present an enhanced recovery after a period of rest. Although the zebrafish larva offers important experimental advantages, experiments in this system are restricted to basic learning paradigms. Because of this, a part of this thesis is devoted to complex visual learning in the Ambon damselfish. This fish is particularly skilled at visual discrimination tasks at it uses facial patterns to discriminate among individuals and other similar species (Siebeck, 2004; Siebeck et al., 2010). Using operant conditioning methods to train the fish for visual discrimination tasks and immunohistological techniques, I attempted to localize the possible forebrain areas involved in visual learning. Exploratory results suggest the involvement of parts of the telencephalon during these tasks. Finally, I attempted to disrupt their visual learning performance while simulating an ocean acidification scenario, as it has been shown to have sensory detrimental consequences in fish (Clements and Hunt, 2015; Nagelkerken and Munday, 2016). However, my results showed an absence of notable differences in visual discrimination and visual learning, which suggest that adult Ambon damselfish visual abilities were not affected. The results of this thesis identified multiple teleost brain structures involved in visual learning, many of which are homologues of subcortical mammalian pathways. The relevance of these findings, including in relation to the human nervous system, is addressed in the final discussion of the thesis. Altogether, these results are important to better understand vertebrate’s visual learning and they open future directions to further investigate visual cognition in teleosts. II Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. III Publications included in this thesis – Marquez-Legorreta E, Piber M, and Scott EK. (2019) Visual Escape in Larval Zebrafish: Stimuli, Circuits, and Behavior. In: The behaviour genetics of zebrafish (Danio rerio). Elsevier. Submitted manuscripts included in this thesis – Marquez-Legorreta E, Constantin L, Piber M, Favre-Bulle IA, Taylor MA, Vanwalleghem GC, and Scott EK (submitted. Under review in Nature) Brain-wide visual habituation networks in wild-type and fmr1 zebrafish. Other publications during candidature Oral presentations – Marquez-Legorreta E, Constantin L, Piber M, Favre-Bulle I, Taylor M, Vanwalleghem G, Scott E. Brain-wide visual habituation networks in wild type and fmr1 zebrafish. Annual Conference of the Australasian Neuroscience Society, Adelaide, 2019. – Marquez-Legorreta E, Constantin L, Piber M, Favre-Bulle I, Taylor M, Vanwalleghem G, Scott E. Brain-wide visual habituation networks in wild type and fmr1 zebrafish. Zebrafish Neural Circuit and Behaviour, Cold Spring Harbor Laboratories, 2019. – Marquez-Legorreta E, Favre-Bulle I, Taylor M, Heap, L, Vanwalleghem G, Scott E. Habituation to looming stimuli in zebrafish larvae. 38th Meeting of the Australasian Neuroscience Society, 2018. – Marquez-Legorreta E, Favre-Bulle I, Taylor M, Heap, L, Vanwalleghem G, Scott E. Habituation to looming stimuli in zebrafish larvae. 9th SBMS International Biomedical Postgraduate Symposium. The University of Queensland, 2018. – Marquez-Legorreta E, Favre-Bulle I, Taylor M, Heap, L, Vanwalleghem G, Scott E. Habituation to looming stimuli in zebrafish larvae. 19th Australia and New Zealand Zebrafish Meeting, Wollongong, Australia, 2018. – Marquez-Legorreta E, Bender D, Parker AN, Rosinski C, Siebeck UE. Visual learning in a changing climate. Heron Island Research Station Research Seminars. 9th of June 2017. – Marquez-Legorreta E, Bender D, Parker AN, Rosinski C, Siebeck UE. Fish visual discrimination and learning in a changing world. 43rd Annual Conference of the Australasian Society for the Study of Animal Behaviour. July of 2016. IV Poster presentations – Marquez-Legorreta E, Favre-Bulle I, Taylor M, Heap, L, Vanwalleghem G, Scott E. Habituation to looming stimuli in zebrafish larvae. International Congress of Neuroethology, Brisbane, Australia, 2018. – Marquez-Legorreta E, Favre-Bulle I, Taylor M, Heap, L, Vanwalleghem G, Scott E. Zebrafish larvae habituation to looming stimuli. 8th SBMS International Biomedical Postgraduate