UNIVERSIDADE DE LISBOA FACULDADE DE MEDICINA

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1 UNIVERSIDADE DE LISBOA FACULDADE DE MEDICINA Neural Mechanisms of Social Cognition in Zebrafish Leonor Inácio Fragata Carreira Orientadores: Professor Doutor Rui Filipe Pais de Oliveira Professora Doutora Ana Maria Sebastião Dissertação especialmente elaborada para obtenção do grau de Mestre em Neurociências 2017

2 "A impressão desta dissertação foi aprovada pelo Conselho Científico da Faculdade de Medicina de Lisboa em reunião de 18 de Julho de ii

3 UNIVERSIDADE DE LISBOA FACULDADE DE MEDICINA Neural Mechanisms of Social Cognition in Zebrafish Leonor Inácio Fragata Carreira Orientadores: Professor Doutor Rui Filipe Pais de Oliveira Professora Doutora Ana Maria Sebastião Dissertação especialmente elaborada para obtenção do grau de Mestre em Neurociências 2017 iii

4 Funding: BIAL 339/14 iv

5 ACKNOWLEDGMENTS Em primeiro lugar, gostaria de agradecer às instituições que me acolheram e graças às quais estou agora a terminar o mestrado numa área que tanto me fascina, à Faculdade de Medicina de Lisboa e ao Instituto Gulbenkian de Ciência. Um agradecimento também à Fundação Bial por financiar este projecto e sem a qual esta tese não seria possível de realizar. Ao Professor Doutor Rui Oliveira pela oportunidade que me deu ao acolher-me no seu grupo e pelas opiniões e discussões sempre presentes que permitiram melhorar este trabalho. À Professora Doutora Ana Sebastião por ter aceite co-orientar este projecto e também por me ter acolhido no seu laboratório quando ainda estava a terminar a minha licenciatura. À Professora Doutora Maria José Diógenes (Mizé) por me ter permitido o meu primeiro contacto a sério com as Neurociências e por todos os conhecimentos que me transmitiu neste primeiro estágio e, mais tarde, enquanto professora no mestrado. Um agradecimento muito especial à Rita Nunes, por teres tornado todo este projecto de tese possível, por tudo o que me ensinaste neste último ano e meio mas, mais do que isso, pela amizade, pela paciência e por seres a melhor "mini-chefe" que eu poderia pedir. Sem a tua ajuda nada disto teria sido possível. Mais um agradecimento particular, à Júlia, por todas as vezes que paraste de fazer experiências para me salvares das in situ e por tudo o que me ensinaste e voltaste a ensinar quando eu já não me lembrava. E, claro, por todas as histórias e risadas na bancada. Aos restantes membros do IBBG: Magda, Sara, Daniela, Ibukun, Felipe, Diogo e António (espero não me estar a esquecer de ninguém) por me fazerem sentir em casa mesmo quando passamos o dia no laboratório, por todas as discussões produtivas mas, ainda mais, por todos os almoços, lanches, retiros e risos. Aos membros da Fish Facility e da Histologia por toda a ajuda e paciência ao longo deste ano e meio. Às amigas de sempre que são para sempre: Ana Duarte, Carolina Paiva e Carolina Collinge. Mesmo que os cafés sejam difíceis de combinar, vocês estão sempre lá como têm estado nos últimos anos. Não preciso de dizer mais, vocês já o sabem. Às pessoas que me mostraram que no mestrado também se fazem amigos para a vida: Ana Drumond, Cátia Alves e Joana Carvalho. Este mestrado foi muito melhor graças a vocês, a todas as conversas, a todos os jantares (e quase-jantares), a todas as vezes em que era demasiado difícil estar atenta nas aulas. Obrigada por fazerem parte desta etapa tão importante. Aos amigos que não estão sempre ao pé de nós mas que estão sempre connosco e cuja amizade não muda nunca: Tiago Pedreira e Diogo Fortunato. Sei que sou uma chata mas obrigada por serem sempre os mesmos e por saber que posso contar com vocês. Ao Miguel, obrigada por tudo. É difícil arranjar palavras para descrever o quão importante foste para mim nesta etapa (e durante os últimos anos!), mas obrigada por seres o meu refúgio da realidade. Obrigada por estares sempre presente e me apoiares em tudo, por me ouvires e me aturares. O resto tu já sabes. v

6 Para os meus papás, porque vocês é que tornaram possível todo o meu percurso até aqui. Obrigada por acreditarem sempre em mim e por me deixarem seguir os meus sonhos e ser cientista. Sem vocês não seria o que sou hoje e nunca poderei agradecer-vos o suficiente. E a ti maninho, por todas as vezes que me perguntaste para que é que o meu trabalho servia e por todas as discussões sobre que ciência é a melhor. E, claro, por seres o meu parceiro no crime. Agora, faz melhor! vi

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8 TABLE OF CONTENTS List of abbreviations... xi List of figures... xiii List of tables... xv Resumo... xvi Abstract... xix 1. Introduction Social Cognition and the Social Brain Nonapeptides: oxytocin as key regulator of social behaviour Zebrafish as a model organism Animacy perception: a particular case of social cognition Unveiling neuronal circuits/mechanisms in zebrafish Objectives Chapter 1: Effect of social and non-social stimuli on zebrafish behaviour Material and Methods Animal housing Behavioural paradigm Video analysis Statistical analysis Results Chapter 2: Animacy detection in zebrafish Validation of adult zebrafish behavioural response to video playbacks Material and Methods viii

9 Animal housing Behavioural paradigm Video analysis Statistical analysis Results Animacy perception in zebrafish: detection of acceleration cues Material and Methods Animal housing Behavioural paradigm Video Analysis Statistical analysis Results Chapter 3: Involvement of oxytocin-like peptides in modulating adult zebrafish response towards acceleration cues Material and Methods Animal housing and genetic screening Behavioural paradigm Video analysis Statistical analysis Results Chapter 4: Characterization of the neural circuitry underlying animacy perception (c-fos activity) Material and Methods Animal housing Behavioural paradigm ix

10 Sampling In situ hybridization Obtaining plasmidic DNA and probe synthesis In situ hybridization in slices Immunohistochemistry Results Discussion Conclusions References Supplementary Material x

11 LIST OF ABBREVIATIONS ANOVA BCIP Botx BSA DAPI DIG Dm EDTA GFP GPCR HINGS IEGs IHC IRs ISH IT KO MgCl 2 MT NaCl NaOH NBT NPO OXT PBS PCIA PCR PFA PLD PTZ Analysis of variance 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt Botoxulin Bovine serum albumin 4',6-diamidino-2-phenylindole Digoxigenin Dorsal nucleus of the ventral telencephalic area Ethylenediaminetetraacetic acid Green fluorescent protein G-protein coupled receptor Heat-inactivated goat serum Immediate-early genes Immunohistochemistry Infra-reds In situ hybridization Isotocin Knockout Magnesium chloride Mesotocin Sodium chloride Sodium hydroxide Nitro-blue tetrazolium chloride Neurosecretory preoptic area Oxytocin Phosphate-buffered saline Phenol/chloroform/isoamyl alcohol Polymerase chain reaction Paraphormaldehyde Point-light displays Pentylenetetrazole xi

12 ROI SDS SEM SSC TBST TL UAS Vd VP VT Vv WT Region of interest Sodium dodecyl sulfate Standard error of the mean Saline-sodium citrate Tris-buffered saline tween Tupfel long fin Upstream activator sequence Ventral nucleus of the dorsal telencephalic area Vasopressin Vasotocin Ventral nucleus of the ventral telencephalic area Wild type xii

13 LIST OF FIGURES Figure 1. Oxytocin and vasopressin homologues through taxa (Donaldson et al., 2008)... 2 Figure 2. Shoaling behaviour in the zebrafish... 4 Figure 3. Upper panel: sequential frames from a dynamic display of the type used by Heider and Simme to demonstrate perceptual animacy - the large triangle might be seen as wanting to catch the small triangle. Lower panel: examples of some of Michotte s basic demonstrations of perceptual causality - the launching effect... 5 Figure 4. Examples of different point-light stimuli. Upper panel: human (left) and pigeon (right). Adapted from Troje et al., Lower panel: fish (medaka fish). Adapted from Nakayasu et al., Figure 5. Schematic representation of the experimental setup Figure 6. Schematic representation of the timeline used in the behavioural protocol Figure 7. Effect of social and non-social stimuli on zebrafish behaviour Figure 8. Effect of social and non-social stimuli on the locomotor activity of the focal fish Figure 9. Schematic representation of the behavioural setup. Stimuli (video playbacks) are presented in two LCD screens, placed in each side of the experimental tank Figure 10. Representative frame of the stimuli (video playbacks) used Figure 11. Validation of adult zebrafish behavioural response to video playbacks Figure 12. Validation of adult zebrafish behavioural response to video playbacks (shoal versus single fish) Figure 13. Schematic representation of the stimuli presented: inanimate versus animated dot. Stimuli are presented in two LCD screens, placed in each side of the experimental tank. 22 Figure 14. Adult zebrafish ability to detect acceleration cues (speed-changes) in a single object Figure 15. Left panel: Dorsal view of zebrafish larvae with isotocin neurons expressing BotxBLC-GFP. Right panel: Close up view of the isotocinergic neurons Figure 16. Sequencing results - chromatogram for each of the three possible genotypes for the IT receptor KO line: wild type (+,+), heterozygotes (+,-),and KO (-,-) fish Figure 17. Involvement of isotocin in modulating adult zebrafish response towards acceleration cues in a transgenic line with impaired isotocin vesicular release xiii

14 Figure 18. Involvement of isotocin in modulating adult zebrafish preference towards acceleration cues in a transgenic line with impaired isotocin vesicular release. Comparison between the preference score, regarding the sex and the genotype Figure 19. Involvement of isotocin in modulating adult zebrafish response towards acceleration cues in an isotocin receptor mutant line Figure 20. Involvement of isotocin in modulating adult zebrafish preference towards acceleration cues in an OXTr mutant line. Comparison between the preference score, regarding the sex and the genotype Figure 21. In situ hybridization of c-fos reveals neural activity when fish are exposed to specific visual stimuli Figure 22. Mapping of c-fos neural activity. c-fos positive cells are shown in dark blue 40 Figure 23. In situ hybridization of c-fos reveals neural activity when fish are exposed to PTZ (positive control) Figure 24. Immunohistochemistry for c-fos activity in Dm (medial zone of the dorsal telencephalic area) Figure 25. Immunohistochemistry for c-fos activity in Vv (ventral nucleus of the ventral telencephalic area) xiv

15 LIST OF TABLES Table 1. Effect of sex and genotype (WT or OXT, UAS:BOTxBLC) on the preference score towards the animated stimulus. Significant effect of sex (F(1,55), p<0.05) and genotype (F(1,55), p<0.01). No significant interaction between sex and genotype. Two-way ANOVA 32 Table 2. Effect of sex and genotype (WT or OXTr KO) on the preference score towards the animated stimulus. Significant effect of sex (F(1,55), p<0.001). No significant effect of the genotype and no significant interaction between sex and genotype. Two-way ANOVA xv

16 RESUMO De forma a poderem sobreviver e reproduzir-se, os animais que vivem em grupo devem cooperar e competir entre si. Estudos prévios sugerem que habitar nestes ambientes sociais elaborados conduz à evolução de estruturas cerebrais executivas de maiores dimensões devido a exigências cognitivas mais elevadas. Contudo, esta hipótese parece ser demasiado simplista, uma vez que, animais que possuem encéfalos de pequenas dimensões (como, por exemplo, abelhas ou peixes-zebra) apresentam comportamentos sociais complexos. Como tal, é necessária uma abordagem alternativa que não se centre na dimensão do encéfalo em si, mas que se foque nos circuitos neuronais subjacentes à cognição e ao comportamento. De facto, foi já descrita em vertebrados uma rede neuronal partilhada por inúmeros comportamentos sociais, baseando-se em padrões conservados de expressão génica e de sistemas neuroquímicos. Esta rede é regulada por neuromoduladores, incluindo a família dos nonapéptidos que tem sido, por sua vez, extensivamente associada à regulação de diversos comportamentos sociais. Esta família, conservada entre os vertebrados, é constituída pela oxitocina e os homólogos mesotocina (aves e répteis) e isotocina (peixes) e também pela vasopressina e o seu homólogo em espécies não-mamíferas, a vasotocina. Tanto a oxitocina como a vasopressina têm a capacidade de se ligar não só ao seu próprio receptor, como também ao receptor do outro nonapéptido, tornando-se difícil a interpretação dos resultados derivados de manipulações farmacológicas e, portanto, este método não permite compreender na totalidade a função destes dois neuromoduladores no comportamento social. Este facto destaca a importância de integrar esta metodologia com estudos mais detalhados recorrendo, por exemplo, a uma espécie social para a qual se encontram já disponíveis ferramentas genéticas que permitam manipular o ganho e a perda de função das populações neuronais da família da oxitocina. É neste contexto que se destaca o peixe-zebra como modelo de estudo. O peixe-zebra é uma espécie altamente social que vive em cardumes com relações sociais bem estruturadas em termos de dominância hierárquica, apresentando comportamentos de agressividade e de corte já caracterizado. Para além disso, outras capacidades cognitivas sociais, como a aprendizagem social e o reconhecimento social foram já descritas nesta espécie. Tendo em conta todas estas características, a investigação da cognição social no peixezebra pode providenciar informação relevante acerca da complexidade do circuito neuronal necessário para uma determinada capacidade, sobretudo se for uma aptidão cognitiva social baseada na capacidade visual, como a percepção de animacidade. A percepção de animacidade é um dos aspectos fundamentais dos processos cognitivos sociais que permite diferenciar um organismo vivo de outros objectos no ambiente sendo, por isso, essencial para a sobrevivência e para a reprodução. Actualmente, já se tem conhecimento de que o sistema visual humano é capaz de detectar animacidade através da observação de pontos de luz colocados nas articulações principais de uma figura humana. De facto, até simples padrões visuais, como formas geométricas em movimento, podem dar originar percepção de animacidade, sobretudo se na sua trajectória apresentarem xvi

17 alterações na velocidade (acelerações) ou mudanças de direcção. Para além disto, estudos indicam a possibilidade de existir um mecanismo para a detecção de animacidade conservado evolutivamente em diferentes espécies (chimpanzés; galinhas; peixe Medaka) e é, provavelmente, inato. Estas duas condições constituem uma oportunidade única de testar se esta capacidade cognitiva é regulada por neuromoduladores e de caracterizar os mecanismos neuronais que lhe estão subjacentes através da identificação das regiões cerebrais activadas durante este processo recorrendo, por exemplo, a ensaios de hibridação in situ ou de imunohistoquímica para marcadores de actividade neuronal, como o c-fos. De modo a compreender que características específicas do movimento de um organismo vivo atraem a atenção do peixe-zebra, neste projecto focámo-nos numa única característica da animacidade a aceleração. Assim, recorrendo à apresentação de vídeos, os indivíduos foram expostos a estímulos simples representados por um círculo preto que se movia num fundo homogéneo. Este estímulo poderia apresentar uma velocidade constante durante todo o seu percurso (estímulo não animado) ou poderia apresentar uma alteração na velocidade (aceleração) a meio da sua trajectória (estímulo animado). Para além disto, com o propósito de determinar se a isotocina desempenha um papel nesta função cognitiva, foram ainda testados na mesma tarefa comportamental linhas transgénicas e mutantes que apresentam défices no sistema isotocinérgico. Assim, e considerando que os ecrãs utilizados para a apresentação dos estímulos são apenas adaptados ao sistema visual humano foi necessário validar a utilização de vídeos em peixe-zebra, recorrendo a um comportamento já extensivamente descrito nesta espécie: a preferência para se aproximarem de um conspecífico ou de um cardume de conspecíficos. Após a optimização do protocolo experimental e dos estímulos visuais a ser utilizados demonstrámos, pela primeira vez, que o peixe-zebra é capaz de detectar animacidade através de pistas de aceleração e que existe, de facto, uma maior motivação por parte dos indivíduos para se aproximarem do estímulo animado. É importante referir que estudos futuros deverão focar-se noutro tipo de pistas de animacidade, como mudanças de direcção, de forma a caracterizar melhor esta preferência. O presente estudo parece ainda ilustrar diferenças entre o modo como os machos e as fêmeas captam estes indicadores de animacidade: nos machos a preferência para o estímulo animado é mais evidente nos dois primeiros minutos do teste e nas fêmeas esta preferência mantém-se constante durante todo o teste. Seria de esperar que défices no sistema isotocinérgico afectassem a capacidade de detectar animacidade. No entanto, contrariamente ao previsto, não se verificou uma perda da preferência pelo estímulo animado em nenhuma das duas linhas testadas. De facto, enquanto os machos wild type exploraram o estímulo animado sobretudo nos dois primeiros minutos do teste, os machos transgénicos e mutantes exibem esta preferência durante todo o período de teste. Relativamente às fêmeas, não foram observadas diferenças comportamentais entre o grupo wild type e os grupos geneticamente modificados, sugerindo que a isotocina apenas modela a percepção de animacidade nos machos. xvii

18 Resultados preliminares demonstram que o estímulo animado (com aceleração) parece induzir a activação de regiões cerebrais como o núcleo ventral da área telencefálica ventral (septo lateral nos mamíferos), região já associada ao comportamento social em diversos estudos mas, também, regiões como o núcleo dorsal da área telencefálica ventral e a zona média da área dorsal telencefálica. Contudo, não é possível ainda retirar conclusões relativas às regiões que são especificamente activadas durante a percepção de animacidade dado que estas experiências se encontram ainda a decorrer e os exemplos apresentados neste projecto não permitem destacar diferenças entre os dois estímulos testados. É então fundamental determinar este padrão de activação neuronal para compreender os mecanismos subjacentes a esta capacidade cognitiva social tão fundamental para a sobrevivência. Para além disto, seria também relevante confirmar que não existe de facto nenhuma via de sinalização a ser activada nos indivíduos transgénicos e mutantes examinando, por exemplo, a possível co-localização entre os receptores de isotocina e o marcador de activação neuronal, c- fos. Palavras-chave: comportamento social; percepção de animacidade; isotocina; peixe-zebra. xviii

19 ABSTRACT In order to survive and reproduce, group-living animals must compete and cooperate with others. According to earlier studies, these complex social environments lead to the evolution of larger executive brain structures. However, this perspective might be too simplistic since small-brained animals, as bees and fish, also present highly complex social behaviours. Therefore, it is necessary an alternative approach that focus in the neural circuits underlying cognition and behaviour rather than focusing on the brain size. In fact, it has already been described a common neural network shared by several social behaviours and possibly regulated by neuromodulators, including the nonapeptide family: oxytocin and vasopressin, which are conserved across vertebrate taxa. As a highly social species with a genetic toolbox available that allows the manipulation of the OXT-like neuronal populations, the zebrafish model organism represents the ideal model to investigate social cognition and to understand the complexity of the neural circuitry required for a given function, particularly if it is a vision-based social cognitive ability, such as animacy perception. Animacy perception is one of the fundamental aspects of social-cognitive processes that can help differentiate living organisms from other objects in the environment, being essential for survival and for reproduction. An evolutionary conserved mechanism for animacy detection seems to be present across different species, which offers a unique opportunity to test if this cognitive ability is regulated by neuromodulators and to characterize its underlying neural mechanisms, through the identification of the brain regions activated during this process. Using a two-choice test and video playbacks as a tool to present the stimuli, we have been able to demonstrate, for the first time, that adult zebrafish can detect animacy based on acceleration cues and that these individuals are more motivated to approach this animated stimulus. Additionally, this study seems to illustrate differences between how males and females perceive animacy cues and the data obtained for the transgenic and the mutant lines suggests that isotocin only modulates the perception of animacy cues in male fish. Future studies should address other animacy cues, such as changes in direction, in order to disentangle which specific motion features drive individuals' attention. Furthermore, the previously mentioned findings highlight the need of determining which brain regions are being activated during animacy perception, in order to disentangle the mechanisms underlying this social-cognitive ability that is essential for survival. Keywords: social behaviour; animacy perception; isotocin; zebrafish. xix

20 1. INTRODUCTION 1.1. Social Cognition and the Social Brain In order to survive and reproduce, group-living animals must compete and cooperate with others. Therefore, natural selection has favoured individuals that are equipped with the cognitive architecture to navigate a social world in which they must make rapid decisions about when and whether to involve themselves in a given social interaction (Platt et al., 2016). Earlier studies suggested that living in a complex social environment leads to the evolution of larger executive brain structures in order to accommodate the increase in computational power due to higher cognitive demands (Dunbar, 2017; Adolphs, 2009). However, recent works suggest that this hypothesis (so called social brain hypothesis) may be too simplistic. For example, it has been showed that small-brained animals, such as bees and fish, present highly complex social behaviours (Chittka et al., 2009; Lihoreau et al., 2012; Oliveira et al., 2011; Shultz et al., 2007).These and other recent findings suggest that big brains are not always needed for complex abilities, and so, instead of the use of brain size, one should analyze the neuronal circuits underlying cognition and behaviour and how those maintain evolutionarily conserved across species. In accordance with this view, an evolutionary conserved social decision-making network (englobing the social behaviour network and the mesolimbic reward system) (O Connell et al., 2011) has been described across vertebrates, based on conserved patterns of gene expression and neurochemical systems (O Connell et al., 2012). This decision-making network is modulated by neuropeptides/neurotransmitters including the nonapeptide family, which are conserved across vertebrate taxa ((Goodson et al., 2011). Their receptors are expressed in the six nodes of the social behaviour network (Huffman et al., 2012; O Connell et al., 2012) and there is extensive literature supporting their role in the regulation of social behaviour among taxa (Reddon et al., 2015) Nonapeptides: oxytocin as key regulator of social behaviour The vertebrate nonapeptides are all derived from arginine vasotocin (VT), and the duplication of this gene in early-jawed fish gave rise to separate clades, which include the mammalian peptides arginine vasopressin (VP) and oxytocin (OXT) (Goodson et al., 2011). Arginine VP was derived from a single amino acid substitution in mammals, whereas VT is present in all non-mammalian species examined so far ((Hoyle, 1998). On the other hand, the oxytocic peptide lineage emerged due to a gene duplication event in early fish temporally associated with two amino acid substitutions in the duplicated gene product (Goodson, 2013). The OXT-like peptides include isotocin (IT) in bony fish and mesotocin (MT) in lungfish, amphibians, reptiles, birds and some marsupial mammal species (Figure 1) (Goodson et al., 2010). 1

21 Figure 1. Oxytocin and vasopressin homologues through taxa. From Donaldson et al., 2008 Oxytocin, specifically, is synthesized in the supraoptic and paraventricular nuclei of the hypothalamus in mammals as an inactive precursor protein, prepro-oxytocin-neurophysin I (Mitre et al., 2016; Shojo et al., 2000). This inactive prohormone is subject to a posttranslational progressive hydrolysis to originate the active form of OXT. Oxytocin is then released into the blood by the posterior pituitary lobe, mediating the described classical roles of OXT, such as milk-ejection reflex and uterine contractility regulation. More recently, it has been shown that oxytocin neurons also project centrally, suggesting that oxytocin acts in the brain to, most likely, regulate social behaviours. Several works support this hypothesis, showing that in several species, a pharmacological manipulation of OXT modulates social cognition and affiliative behaviour in both males and females across taxa (Ross et al., 2009). In mammals, OXT modulates social recognition, mother-infant bonding and pair bonding behaviours in monogamous species (Johnson et al., 2016). In birds, the OXT homologue mesotocin stimulates preferences for larger groups and familiar same-sex stimuli in some species (e.g. zebra finches) (Goodson et al., 2010). In fish, IT is known to promote social approach towards conspecifics in species such as zebrafish (Almeida et al., 2011). In other fish, this peptide has effects on socially induced sex changes, breeding vocalizations, and courtship behaviours (Goodson et al., 2000; Thompson et al., 2004; Reddon et al., 2015). The effects of oxytocin are mediated by the oxytocin receptor (OXTR), a G-protein coupled receptor (GPCR). While in mammals there is only one isoform for the receptor, in teleost fish occurred a duplication of the gene originating two receptor isoforms (oxytocin receptor and oxytocin-like receptor), but those are still not very well characterized (Wircer et al., 2016). Although with lower affinity, oxytocin can also bind to vasopressin receptors, and VP can also bind to OXT receptors, in both mammalian and non-mammalian species (Kelly et al., 2

22 2014). This cross-reactivity makes it difficult to interpret data from pharmacological manipulations: even the most adequate antagonist available will, at higher concentrations, cross-react with the receptor of the other related nonapeptides (vasopressin). Thus, it is difficult to dissect the roles of these two neuropeptides in social behaviour (Englemann et al., 2000). Therefore, this approach should be integrated with more detailed studies using tools that allow a higher specificity in order to investigate their particular roles on social behaviour. This aim could be achieved by the use of simpler-minded but still highly social species, for which genetic tools are available to genetically manipulate the gain and loss of function of the OXT-like neuronal populations; this is the case of zebrafish Zebrafish as a model organism Different model organisms have been extensively used to study social cognitions, especially since the development of genetically based tools for studying the activity, anatomy and function of neural circuits (Anderson et al., 2014). Rodents (rats and mice) are the most common animal models used. However, simpler systems as nematodes, flies and bees can also be very useful in the study of social cognition because they share many common behaviours with more complex animals and their genomes and nervous systems are easy to manipulate (Sokolowski, 2010). Moreover, other non-mammalian and more evolved organisms, such as zebrafish (Danio rerio), have emerged in this field (Abril-de-Abreu et al., 2015; Teles et al., 2016; Al-imari et al., 2008). In the past years, a full repertoire of zebrafish social behaviours have been characterized by different research groups, making this species a golden model to study social cognition. When compared to classic invertebrate genetic model organisms (e.g. Drosophila melanogaster, Caenorhabditis elegans), zebrafish is a vertebrate and therefore is more closely related to humans. In addition, when compared to other vertebrate systems such as rodents, zebrafish are smaller (3 4 cm long when they are adults), have a similar generation time (3 months) but from each cross, zebrafish females can lay several hundred eggs, and therefore a large number of animals can be easily maintained in a relatively small space, which is an advantage for large-scale research. Zebrafish embryos are transparent and develop externally allowing for early genetic manipulation (Oliveira, 2013). Zebrafish is a highly social species. In nature and in the lab, zebrafish individuals swim closely together, forming mixed-sex shoals (Figure 2) (Spence et al., 2008). Shoals with multiple individuals may detect predators sooner and confuse them, since it is more difficult to focus on groups than on a single target fish. Besides its role on anti-predator defenses, shoaling behaviour may also facilitate foraging efficiency and mating success (Gerlai et al., 2014). Each individual can shoal with another single individual or with a large group, however the association with a single fish is of lower magnitude. If fish prefer to shoal with a single individual over a larger group, this social preference is likely mediated by factors associated with social recognition, aggression or mating (Fernandes et al., 2015). Zebrafish also display a strong social preference to associate with conspecifics. In the laboratory, this affiliative behaviour can be studied using a test called social/shoal preference test. This test measures the motivation of the zebrafish to approach conspecifics (either real ones or their computeranimated images) in a two-choice preference test paradigm. In this behavioural paradigm, the 3

23 time spent close to the stimulus fish (or groups of fish) is recorded and taken as a measure of preference (Wright et al., 2006). Despite the affiliative behaviours above mentioned, zebrafish display other functional social behaviours, such as aggression, dominance hierarchies (Oliveira et al., 2011), and matechoice behaviours with a well-characterized courtship behaviour (Darrow et al., 2004). Moreover, other basic social-cognitive processes have already been described in the zebrafish, including the ability to collect information from others (social attention); recognize conspecifics (social recognition); and learn from and about others (social learning) (Oliveira, 2013). These complex behaviours can be regulated by neuromodulators. As mentioned before, an obvious candidate is IT. In zebrafish, IT is produced in the neurosecretory preoptic area (NPO), and, similar to what happens in mammals, it is released into the blood via neurohypophysis or is released in different brain areas where it may regulate social behaviours, since there are already evidences in this species showing an implicating of IT in social behaviour and fear response to predator (Braida et al., 2012; Almeida et al., 2012). Figure 2. Shoaling behaviour in the zebrafish. Considering all the features mentioned above, the investigation of social cognition in the zebrafish can provide information about the complexity of the neural circuitry required for a given function (Salva et al., 2014), particularly if it is a vision-based social cognitive ability, since the visual capabilities of fish are highly developed and that many fish species perceive their world largely through the visual system (Schluessel et al., 2015). 4

24 1.4. Animacy perception: a particular case of social cognition It has been hypothesized that the mechanisms controlling an individual s interactions with other behavioural agents differ from those involved in the interactions of this individual with its physical environment. Therefore, in order to consider these putatively different mechanisms, a new concept emerged: social cognition. Social cognition comprises cognitive processes that are specifically used in social behaviour, such as recognition of individuals or social categories, social partner preferences, development and management of social relationships, social learning, social coordination, manipulation and punishment (Oliveira et al., 2013; Menzel et al., 2011). Although individuals exhibit a broad social behaviour repertoire, it is important to notice that in all of these cognitive processes it is fundamental to differentiate living organisms from other objects in the environment and this relies on to the ability to perceive animacy, which can be defined as the property of being alive. Thus, one fundamental property of the social brain involves systems for recognizing and understanding animate agents (Gobbini et al., 2011). The identification of other animate entities is essential for survival, since it allows the detection of potential predators or prey in the visual field and for reproduction, since it allows to recognize potential mates. (Morito et al., 2009; Lowder et al., 2015; Thurman et al., 2014). In nature, most relevant stimuli such as those elicited by conspecifics involve movement. Thus, perceiving motion should be ecologically relevant and should have been selected for (Schluessel et al., 2015). For cognition in general, animate stimuli capture visual attention more quickly and hold attention longer than inanimate stimuli. (Johansson, 1973; Pratt et al, 2010). Heider and Simmel (1944) followed by Michotte (1963) were the first to demonstrate that animations of simple geometric shapes interacting with one another can elicit the perception of animacy based on how these shapes move together. These pioneering works suggested that just as the visual system works to recover the physical structure of the world by inferring properties as 3-D shape, it is also capable of recovering social structure of the world by inferring properties such as causality and animacy, even with visual displays containing only small-moving 2-D geometric shapes (Figure 3) (Heider et al. 1944; Michotte, 1963). Figure 3. Upper panel: sequential frames from a dynamic display of the type used by Heider and Simme to demonstrate perceptual animacy - the large triangle might be seen as wanting to catch the small triangle. Lower panel: examples of some of Michotte s basic demonstrations of perceptual causality - the launching effect. 5

25 One of the most well studied visual cues that induce spontaneous animacy perception in human adults is biological motion (Pavlova, 2012). By definition, biological motion refers to the characteristic, non-rigid patterns produced when humans or other animals move constrained by their skeletal structure (Poulin-dubois et al., 2015). Perception of biological motion has been studied by presenting point-light displays (PLD) that prevent the use of twodimensional information about the shape of the moving object (Johansson, 1973). These stimuli consist of an animation sequence obtained by placing few light points on the joints of a digitalized image of a moving animal. It is already known that the human visual system can detect the presence of a human when looking at dozen of point lights placed on the main joints of a walking person. In fact, visual preference for biological motion has been demonstrated in newborns, and 3-month-old and 12-month-old infants are able to extract social-cognitive cues, as gaze following, from a point-light display of a human walking (Poulin-dubois et al., 2015). Indeed, the propensity for detecting and analyzing biological movement patterns appears to be deeply ingrained not only in the human brain but also in other vertebrate species, such as, newly-hatched domestic chicks (Vallortigara et al., 2005), pigeons (Troje et al., 2013), monkeys (Oram et al., 1994), and medaka fish (Nakayasu et al., 2014). Figure 4. Examples of different point-light stimuli. Upper panel: human (left) and pigeon (right). Adapted from Troje et al., Lower panel: fish (medaka fish). Adapted from Nakayasu et al.,

26 Nevertheless, in these studies that use biological motion patterns it is still difficult to isolate the motion features essential to the perception of animacy, therefore, studies with simpler visual displays have started to emerge. In fact, simple visual displays like moving geometric shapes (e.g. squares, dots) can give rise to robust percepts of animacy when they move in certain ways. That our visual system is adapted to extract socially relevant information from even such impoverished stimuli demonstrates that elementary motion cues provide the foundation for social perception in general (Gao et al., 2012; Scholl et al., 2000). The advantage of such simple displays is that they permit the study of perceptual animacy without contamination from other factors that might come into play (Pratt et al., 2010). Moreover, with this type of schematic display is easier to control the motion components of the stimuli. Since the first studies from Heider and Simmel (1944) and Michotte (1963), several researchers have tried to disentangle which visual cues of motion promote the perception of animacy in adults, such as speed and trajectory direction changes, discontinuity in motion trajectory, motion contingency (particularly, chasing, in which one visual object appears to be chasing the other (Gao et al., 2009) and violation of Newtonian laws of motion (Di Giorgio et al., 2016). Self-propulsion, which is defined as the capacity to start to move autonomously as opposed to being put in motion by physical contact with another object, is possibly the most widely investigated kinematic cue to animacy. In adults, it has been shown that the onset of motion captures attention more than the continuous motion of an object and even infants are able to attribute an internal energy source to an object that starts to move on its own, expecting it to spontaneously change its own trajectory or to move against gravity (Salva et al., 2015). The important role of self-propulsion as a cue to animacy has also been demonstrated in other species, such as domestic chicken, where newly hatched chicks exposed to digital animation of two objects exhibiting motion either self-produced or caused by physical contact later prefer to approach the self-propelled object (Mascalzoni et al., 2010). In 2000 it was found that animacy was associated with changes in speed (accelerations) and changes in trajectory (in relation to the orientation of the object s main axis) by presenting a single object moving on a homogeneous background. Objects endowed with an internal energy source that accelerate and/or change their motion direction are classified to be more alive and to have higher levels of animacity when compared to an object that needs an external energy source (Tremoulet et al., 2000; Scholl et al., 2000). According to several studies, an evolutionary conserved mechanism for animacy detection, through biological motion patterns or simple geometric shapes, seems to be present across different species. Although, to the best of our knowledge, it has not been described in zebrafish, animacy perception has been observed in other animal models such as chicks (Mascalzoni et al., 2010), pigeons (Goto et al., 2002), Japanese macaques (Tsutsumi et al., 2012), medaka fish (Nakayasu et al., 2014) and rats (MacKinnon et al., 2010). Moreover, developmental studies with newborns and newly hatched chicks support the hypothesis of an innate mechanism. These two conditions offer a unique opportunity to test if this cognitive ability is regulated by neuromodulators, such as the nonapeptide family mentioned above. Furthermore, it is also possible to characterize its underlying neural mechanisms through the identification of the brain regions activated during this process using, for example, in situ hybridization or immunohistochemistry assays for neuronal markers activity. 7

27 1.5. Unveiling neuronal circuits/mechanisms in zebrafish Imediate-early genes (legs) are so called because their transcription is induced rapidly and transiently by many extracellular stimuli, independently of protein synthesis (Cirelli et al., 1995). These IEGs are rapidly induced within activated neurons and their mrna or protein products have long been used as markers of behaviourally activated neurons. These genes can be classified into two categories, regulatory IEGs and effector IEGs. Regulatory IEGs comprise encoding DNA-binding proteins or transcription factors (e.g. c-fos and early growth response protein-1 (egr-1)), whose products modulate downstream target genes. On the other hand, effector IEGs encode proteins that regulate cellular signal transduction more directly than regulatory IEGs do (e.g. activity-regulated cytoskeleton associated protein) (Lee et al., 2016; O Donnell et al., 2012). Since their first neurobiological applications, c-fos and its protein product c-fos became the most widely used markers for identifying activated cells and central nervous system circuits. This happened due to several factors: (i) it is expressed at low levels in the intact brain under basal conditions; (ii) it is induced in response to several extracellular signals; (iii) the response is transient; (iv) detection of c-fos expression, both mrna and protein, is not complicated; (v) it has been suggested that IEG expression in some brain areas under resting conditions is not random, but rather reflects recent experience (Marrone et al., 2008; Luckman et al., 1994; Kovács et al., 2008). Generally, the kinetics of these gene response to acute stimuli is transient, with a peak of c-fos mrna approximately 30 min and c-fos protein between min (Kovács et al., 1998). As a marker for neuronal activation, c-fos and its product c-fos have been extensively used to study the circuits and the brains regions activated during social interactions, resorting to techniques such as in situ hybridization or immunohistochemistry (Sakaguchi et al., 2017; Goodson et al. 2005; Northcutt et al., 2009; Hamilton et al., 2010; Mayer et al., 2017; Malinowska et al., 2016; Ferri et al., 2016). In situ hybridization (ISH) techniques allow specific nucleic acid sequences to be detected in morphologically preserved chromosomes, cells or tissue sections. Early approaches to ISH employed radioactively labeled probes to detect transcripts on histological sections. However, the introduction of nonradioactive labeling systems allowed faster and easier transcript visualization in whole-mounted tissues and embryos. There are two types of non-radioactive hybridization methods: direct (where the detectable/reporter molecule is bound directly to the nucleic acid probe and probe-target hybrids can be visualized under a microscope immediately after the hybridization reaction) and indirect (require the probe to contain a reporter molecule, introduced chemically or enzymatically) (Machluf et al., 2011). Immunohistochemistry (IHC) is a powerful microscopy-based technique for visualizing cellular components, for instance proteins or other macromolecules in tissue samples. The classical IHC assay involves the detection of epitopes expressed by a single protein-target within a tissue sample using a primary antibody capable of binding those epitopes with high specificity. After this event, a secondary antibody capable of binding the primary antibody is added. The secondary antibody is coupled to a reporter molecule and after the antibodyantibody binding event, a chemical substrate is added which reacts with the reporter molecule 8

28 to produce a colored precipitate at the site of the whole epitope-antibody complex (Coons et al., 1941; O Hurley et al., 2014) 9

29 2. OBJECTIVES As previously mentioned, recognition of other behavioural agents in the environment constitutes an elementary cognitive ability for social cognition. In this work we propose to study the underlying neural mechanisms of one specific cognitive ability, the perception of animacy through acceleration cues. The fact that there are evidences for an evolutionary conserved mechanism across different species offers a unique opportunity to test if this competence is regulated by oxytocin-like peptides (IT in fish) - a family of neuromodulators implicated in the regulation of sociality across taxa. The zebrafish model organism represents an ideal model to dissect the questions raised above, since these small-brained vertebrate organisms exhibit a broad social behaviour repertoire. Moreover, due to a complex existing genetic tool box in the zebrafish, current transgenic and mutant lines are already available, allowing a better understanding of the gain/loss of function of OXT-like neurons. Thus, the main aims of this work were: 1) To assess if zebrafish distinguish between social and non-social cues (effect of social and non-social stimuli in zebrafish behaviour), and if so, which particularities does it detect from social cues, more specifically, determine if zebrafish able to detect animacy patterns that characterize living organisms (i.e. acceleration cues); 2) To evaluate if IT plays a role in modulating animacy perception; 3) To determine the neural circuitry underlying this cognitive ability (animacy detection) through the identification of the brain areas activated during this process, using in situ hybridization or immunohistochemistry. To accomplish these aims, the present work was divided in four parts: First chapter: Effect of social and non-social stimuli in zebrafish behaviour. Second chapter: Animacy perception in zebrafish: detection of acceleration cues Third chapter: Evaluation of isotocin role in modulating animacy perception Forth chapter: Characterization of the neural circuitry underlying animacy perception through the identification of the brain areas activated during this process, using in situ hybridization or immunohistochemistry for the neural activity marker cfos. 10

30 3. CHAPTER 1: EFFECT OF SOCIAL AND NON-SOCIAL STIMULI ON ZEBRAFISH BEHAVIOUR As a highly social species, the zebrafish should be able to discriminate between a social or a non-social stimulus. It would be expected that zebrafish would spend more time in association with a conspecific (social stimulus) than with an object (non-social stimulus). In order to evaluate the effect of these two types of stimuli on zebrafish behaviour, subjects were either exposed to a single male conspecific or an object. For the object it was used an eppendorf tube since its fusiform shape is similar to zebrafish s body shape. A third group of fish was exposed to an empty compartment as a control stimulus MATERIAL AND METHODS Animal housing All subjects used in this experiment (n=30) were seven to eight month old male zebrafish from an outcross wild-type Tuebingen strain bred and held at Instituto Gulbenkian de Ciência. Animals were kept in a water recirculation system at 28ºC in a 14h/light: 10h/dark cycle and the water quality was monitored every day: nitrites <0.2ppm, nitrates <50ppm, ammonia ppm, ph = 7 and conductivity at approximately 700 μsm Behavioural paradigm The experimental setup used in this task consisted in a glass tank (30 x 15 x 18 cm) divided in two equally-sized compartments by two partitions: an opaque partition that was removed when the interaction started, and a transparent and perforated one, to allow water flow between the two compartments and also a visual contact between the focal fish and the stimulus (Figure 5). Figure 5. Schematic representation of the experimental setup. Two partitions were placed in the middle of the tank: one transparent partitions allowing both water flow and visual contact and one opaque partition to prevent the focal fish to visualize the stimulus when it is placed in the experimental tank. During the video analysis, a region of interest (ROI) near the stimulus was established. A fixed IR camera recorded the whole 90 s trial from above. 11

31 The focal fish was removed from its home tank and housed individually in 1L glass tanks (11.6 x 11.6 x 15 cm) eight days before the behavioural recording test to increase the effect of subsequent presentation of the stimulus. Fish were then randomly assigned to three different groups: (i) the focal fish were not exposed to any stimulus (control group); (ii) the focal fish were exposed to a conspecific (social stimulus); (iii) the focal fish were exposed to an object - eppendorf tube - (non-social stimulus). Additionally, in order to assess if this long period of social isolation could affect the behavioural response of the focal fish to a conspecific, a fourth group was tested (n=5). In this group the subject was only isolated overnight in the experimental tank and it was exposed to a conspecific in the day after (Figure S1, Supplementary Material). Regarding the object, two distinct colours (red and green) and three different sizes (0.5mL, 1.5mL and 2.0mL eppendorfs) were tested beforehand to optimize the subject s response towards this non-social stimulus (Figure S2, Supplementary Material). Previous works in the literature have showed that these colours do not elicit avoidance in zebrafish (Avdesh et al., 2012). In the day before the experiment, animals were transferred from the isolation tank to one of the compartments of the experimental arena, in order to habituate overnight to the experimental setup. In the day of the experiment the stimulus (either social or non-social) was placed in the other compartment, or the compartment was left empty, and then the opaque partition was lifted and the observation lasted for 90 seconds (Figure 6). After this period, the stimulus (conspecific or object) was removed from the experimental tank using a net. In order to avoid any differences in the fish behaviour associated with the handling of the stimulus, the net was also inserted in the stimulus compartment in the group where no stimulus was presented. The whole experiment was video-recorded for subsequent automated videotracking. Figure 6. Schematic representation of the timeline used in the present behavioural protocol. 12

32 Preference for a stimulus was measured by the time the focal fish spent in the region of interest (ROI) near the stimulus, according to the following equation: Equation 1.1. Preference for a stimulus (%) = Time spent in ROI Time spent in the entire arena Video analysis All behavioural tests were recorded using fixed infra-red (IRs) cameras with an acquisition rate of 30 fps, connected to a laptop using the video recording software, Pinnacle Studio 12 ( The analysis of the behaviour was performed using the video-tracking software Ethovision XT11 from Noldus ( Statistical analysis Due to a small sample size, a Gaussian distribution was not assumed. The present data were analyzed with the nonparametric Mann-Whitney test for unpaired data. Outliers analysis was performed using ROUT test (Q = 1%). Descriptive statistics were presented as mean ± SEM. Statistical significance was set at p < 0.05 (*), p < 0.01 (**), p < (***) and p < (****). All statistical analyses were performed with GraphPad Prism 6 ( 13

33 3.2. RESULTS As described before, focal fish was either exposed to a conspecific fish, an object or an empty compartment for 90 seconds (only one treatment per fish). The object used in this experiment was a red eppendorf tube with a small size (0.5 ml) since these were the object s characteristics that elicited a higher preference and less avoidance, simultaneously (Figure S2, Supplementary Material). Focal fish spent more time in the ROI near to a conspecific fish than near to an object (Figure 7A, p<0.01) or an empty tank (Figure 7A, p<0.01). The heat maps (Figure 7B) show the track/cumulative time of a representative fish of each group during the whole trial: fish exposed to an empty compartment or to an object explored more the whole arena (left and right panels, respectively), while fish exposed to a conspecific spent the entire trial in the ROI (middle panel) (Figure 7B). The colour red reflects the areas where the focal fish spent more time during the trial, whereas the blue one reflects the opposite. Figure 7. Effect of social and non-social stimuli on zebrafish behaviour. A Percentage cumulative time spent near the stimulus in three different treatments: empty tank (Control group), conspecific (Social group) and object (Non-social group). Data is presented as mean ± SEM, ** p<0.01, Mann-Whitney test. B Heat maps from a representative fish for each group displaying the time spent in the different regions of the tank during the whole trial for the three treatments. The area delimited by a yellow line represents the arena established during the video analysis and the area delimited by a red line represents the region of interest (ROI). The regions in red correspond to the zones where the fish spent more time and the regions in blue correspond to the zones where the fish spent less time. 14

34 The mean velocity and total distance covered by the focal fish were assessed in order to evaluate changes in locomotor activity. As observed in Figure 8, exposure to a conspecific fish decreased the locomotor activity of the focal subject comparatively to an exposure to an empty tank (Figure 8, p<0.01) or to an object (trend towards decreased locomotion, p= for Total distance; p= for Mean Velocity). Finally, there was no effect of the long period of isolation used in this experiment (8 days) in the behavioural response of the focal fish towards the presence of a conspecific (Figure S1, Supplementary Material). Figure 8. Effect of social and non-social stimuli on the locomotor activity of the focal fish. Left panel Total distance covered by the focal fish during the whole trial in three different treatments: empty tank (Control group), conspecific (Social group) and object (Non-social group). Right panel Mean velocity of the focal fish during the whole trial in three different treatments: empty tank (Control group), conspecific (Social group) and object (Non-social group). Data is presented as mean ± SEM, ** p<0.01, ns non-significant, Mann-Whitney test. 15

35 4. CHAPTER 2: ANIMACY DETECTION IN ZEBRAFISH 4.1. VALIDATION OF ADULT ZEBRAFISH BEHAVIOURAL RESPONSE TO VIDEO PLAYBACKS Video playbacks allow a standardization of the stimulus presented since they produce more controllable and repeatable visual stimuli than the use of live animals (Qin et al., 2014). However, all video systems are specifically designed for human vision so it was necessary to validate zebrafish s behavioural response to this type of stimulus. For this purpose, we took advantage of a characteristic behaviour of this species: the preference of zebrafish to associate with a shoal of conspecifics. Shoal preference is a highly robust behaviour in zebrafish, and it has been shown that the focal zebrafish displays preference not only for a real shoal but also for a video of shoals: when exposed to a video playback of a shoal of conspecifics, zebrafish spend most of the time in association with the video (Qin et al., 2014; Saverino et al., 2008) MATERIAL AND METHODS Animal housing All subjects used in this experiment (n=45) were five to eight month old male zebrafish from an outcross wild-type TL strain bred and held at Instituto Gulbenkian de Ciência Animals were kept in a water recirculation system,at 28ºC in a 14h/light: 10h/dark cycle and the water quality was monitored every day: nitrites <0.2ppm, nitrates <50ppm, ammonia ppm, ph = 7 and conductivity at approximately 700 μsm Behavioural paradigm The experimental setup used in this task consisted in a glass tank (30 x 15 x 18 cm) divided in three equal compartments: a central area where zebrafish is allowed to habituate to the tank, and two identical outer areas. The stimuli were presented in two LCD screens, placed in each side of the tank and the whole setup was covered by a black cover in order to ensure that there was no other source of light besides the screens (Figure 9). At the beginning of the experiment, the subject was placed in the central compartment for acclimatization, while a video of an empty tank is presented in both screens. After this 10-minute period of habituation to the videos, the stimuli appeared in the screens for more 10 minutes before the partitions were removed and the fish was allowed to explore the entire arena for 20 minutes, while the whole experiment was video-recorded for subsequent video-tracking. In this two-choice test, fish were either exposed to a video of a shoal in one side and a video of an empty tank in the other (n=12) or to a video of a single conspecific opposed to another video of an empty tank (n=12) (Figure 10). 16

36 Figure 9. Schematic representation of the behavioural setup. Stimuli (video playbacks) are presented in two LCD screens, placed in each side of the experimental tank: while one is presenting a video of a shoal or a single fish, the other is presenting an empty tank. The side in which each stimulus was presented was randomized between subjects. The tank is divided in a central area, where fish is allowed to acclimatize, and two identical outer areas, close to the stimuli. Since both the shoal and the single conspecific stimuli were being presented against a video of an empty tank, it could be argued that the salience of the latter stimulus is very different from the other and could bias the subject s preference without meaning that the fish is in fact perceiving the video playbacks. According to the literature, zebrafish prefer a shoal of conspecifics when compared to an empty tank but also when compared to a single fish. Therefore, in order to infer if they could in fact perceive what is being shown in the presented videos, this behavioural preference was also tested using simultaneously in opposite sides, video playbacks of a shoal and single fish. The stimuli was either a shoal of four males presented with a single male conspecifics, or a shoal of four females and a single female conspecific. This combination was performed in order to avoid possible confounding factors by using different sexes in stimuli presented at the same time. Figure 10. Representative frame of the stimuli (video playbacks) used. Left panel: shoal of conspecifics: 2 males and 2 females. Middle panel: single male conspecific. Right panel: empty tank. Videos were recorded using a GoPro Hero 3 camera ( and were edited using the program Adobe After Effects. 17

37 One region of interest (ROI) was defined near each screen and the preference towards a stimulus was measured by the time spent in this region (Equation 1.1.). In order to assess if the preference for the shoal is higher when compared to the single conspecific, a sociality score was calculated based on the following equation: Equation 1.2. Sociality score = Time in target ROI (Shoal or Single Fish) Time in target ROI + Time in empty tank ROI Video analysis All behavioural tests were recorded using fixed infra-red cameras with an acquisition rate of 30 fps connected to a laptop using the video recording software, Pinnacle Studio 12 ( The analysis of the behaviour was performed using the videotracking software Ethovision XT11 from Noldus ( Statistical analysis Due to the small sample size, a Gaussian distribution was not assumed. The data from the present experiment were analyzed with nonparametric tests, namely: the Wilcoxon test for paired data and the Mann-Whitney test for unpaired data. Outliers analysis was performed using ROUT test (Q = 1%). Descriptive statistics were presented as mean ± SEM. Statistical significance was set at p < 0.05 (*), p < 0.01 (**), p < (***) and p < (****). All statistical analysis was performed with GraphPad Prism 6 ( 18

38 RESULTS Based on the percentage of cumulative time spent in the ROI, adult zebrafish exhibited a significant preference towards a shoal of conspecifics, when compared to an empty tank (Figure 11A, p<0.001). The same response pattern was observed when using a single male fish as a social stimulus (Figure 11B, p<0.001). However, the sociality score obtained between shoal as a social stimulus versus empty was significantly higher when compared to the score for a single male stimulus versus empty. Furthermore, an exposure to shoal versus empty elicited a more consistent and robust response than to a single versus empty exposure, during the whole 20 minutes trial (Figure 11C, p<0.001). Figure 11. Validation of adult zebrafish behavioural response to video playbacks. A Percentage cumulative time spent near the stimuli: shoal versus empty tank (non-shoal). B - Percentage cumulative time spent near the stimuli: single fish versus empty tank (non-shoal). Data is presented as mean ± SEM, *** p<0.001, between shoal or single fish versus the empty tank stimulus, Wilcoxon test. C Comparison between the sociality score for a shoal or a single fish stimulus. The sociality score represents the ratio of time spent by the subject in the region of interest near the social stimulus (shoal or single fish) with respect to the time spent in both outer areas near a stimulus (shoal or single fish and non-shoal). Score is presented as a mean value of the 20 min. trial (left panel) and in 1 min. time-bins (right panel). Data is presented as mean ± SEM, *** p<0.001, between sociality scores, Mann-Whitney test. According to the literature zebrafish discriminates between a shoal and a single fish in a two-choice preference test, spending more time in association with the shoal ((Wright et al., 19

39 2006). As observed in Figure 12, when fish were exposed to a video of a shoal (4 males or 4 females) in one side of the tank and a single fish in the opposite side (male or female, depending on the shoal used) (see Methods), they spent most of the time with the shoal, regardless of the sex of the animals. Figure 12. Validation of adult zebrafish behavioural response to video playbacks (shoal versus single fish). A Percentage cumulative time spent near the stimuli: shoal of four male fish versus a single male fish. B - Percentage cumulative time spent near the stimuli: shoal of four female fish versus a single female fish. Data is presented as mean ± SEM, ** p<0.01, *** p<0.001, Wilcoxon test. 20

40 4.2. ANIMACY PERCEPTION IN ZEBRAFISH: DETECTION OF ACCELERATION CUES The fact that zebrafish usually prefer to be near a conspecific or a shoal of conspecifics raises the hypothesis that they prefer social stimuli due to specific features in their motion pattern. It was previously stated that sudden changes in speed (accelerations) can elicit percepts of animacy, even when using simple geometric shapes (e.g. dots) (Scholl et al., 2000). Thus, it was tested if zebrafish would prefer a dot moving with acceleration rather than one moving at constant speed (Figure 13). In this experiment, the stimuli consisted in two black dots moving in the background image previously used in the video playbacks showing a shoal or a single conspecific. The color chosen for the dots was used in order to ensure the maximum contrast between the background and the dots entering the screen from one side and leaving the screen through the opposite side. All these stimuli were generated using Adobe After Effects ( MATERIAL AND METHODS Animal housing All subjects used in this experiment (n=77) were four to eight month old male zebrafish from an outcross wild-type TL strain bred and held at Instituto Gulbenkian de Ciência Animals were kept in a water recirculation system, at 28ºC in a 14h/light: 10h/dark cycle and the water quality was monitored every day: nitrites <0.2ppm, nitrates <50ppm, ammonia ppm, ph = 7 and conductivity at approximately 700 μsm Behavioural paradigm For this task, it was used the behavioural setup previously described (Figure 9) and the same protocol: the focal fish was placed in the central compartment and exposed to a video of an empty tank during 10 minutes for acclimatization; after this period the two stimuli-dots appeared in the screens for more 10 minutes before the partitions were lifted and the fish was allowed to explore the arena for 20 minutes. The experiment was video-recorded for future video-tracking. Similar to the previous experiment, one ROI was defined near each screen and the preference towards a stimulus was measured by the time spent in each of this regions (Equation 1.1). Moreover, it was calculated a preference score towards the animated stimulus: Equation 1.3. Time in target ROI (animated dot) Preference score for acceleration = Time in target ROI + Time in inanimate dot ROI 21

41 First, in order to determine if the size of a dot would affect the zebrafish preference for a single object with changes in speed (animated dot) versus an object with constant speed (inanimate dot), two different dot sizes were tested: a smaller (Ø=0.8 cm, n=9) and a bigger one (Ø=1.6 cm, n=10), being the latest similar with the medium area of an adult zebrafish. Then, to evaluate if mean velocity could influence the subjects response towards the dots two different mean velocities were tested: 0.5 cm/s (n=10) and 2.8 cm/s (n=10). Ideally, a higher mean velocity should have been used to match zebrafish s mean velocity, however this was not possible since the distance covered by each dot was only 15 cm (corresponding to the width of the tank). In order to assess if the long period of habituation inside the central compartment could decrease the interest for one of the stimulus, the same paradigm was tested with only one minute of habituation inside the partitions (n=12). Considering that the response towards these stimuli should be less robust that the response to the shoal/single stimulus, it was important to determine if less predictable trajectories could improve the fish s performance in this task. Therefore, new stimuli were generated: (i) dot moving in a single trajectory but always re-entering the screen from the same side it has left it (back and forward trajectory); (ii) dot moving with multiple trajectories (a schematic representation of this stimulus is shown in Figure S3, Supplementary Material). Figure 13. Schematic representation of the stimuli presented: inanimate versus animated dot. Stimuli are presented in two LCD screens, placed in each side of the experimental tank: while one is presenting a video of a dot moving with a constant speed (left panel), the other is presenting a video of a dot with a speed-change (acceleration) during its trajectory (right panel). The side in which each stimulus was presented was randomized between subjects Video analysis All behavioural tests were recorded using fixed infra-red cameras at a rate of 30 fps, connected to a laptop using the video recording software, Pinnacle Studio 12 ( The analysis of the behaviour was performed using the videotracking software Ethovision XT11 from Noldus ( 22

42 Statistical analysis Due to the small sample size, a Gaussian distribution was not assumed and the data from the present experiment were analyzed with nonparametric tests, namely: the Wilcoxon test for paired data and the Mann-Whitney test for unpaired data. Outliers analysis was performed using ROUT test (Q = 1%). Descriptive statistics were presented as mean ± SEM. Statistical significance was set at p < 0.05 (*), p < 0.01 (**), p < (***) and p < (****). All statistical analyses were performed with GraphPad Prism 6 ( 23

43 4.2.2 RESULTS In both previous experiments the focal fish always chose to be near a conspecific or a group of conspecifics (motion) when compared to an empty compartment or an object (static). Therefore, in order to determine which specific features in their motion pattern were responsible for this preference, it was tested if zebrafish would prefer a dot moving with acceleration rather than one moving at constant speed; in other words, if they could perceive and prefer animacy cues. First, two different dot sizes were tested: a smaller one (Ø=0.8 cm) and a larger one (Ø=1.6 cm) in order to determine if this feature could influence zebrafish s preference towards one of the stimulus. Since no significant differences were observed in the score preference towards the animated dot using these two different sizes (Figure 14A, p = ), the 1.6 diameter dot was used in all future experiments, since it corresponds to the average area of an adult zebrafish. Then, to evaluate if mean velocity could influence the subjects response towards the dots two different mean velocities were tested: 0.5 cm/s and 2.8 cm/s. As shown by Figure 14B, no significant preferences were observed for one of the two stimuli (animated vs inanimate), although there is a tendency for the focal fish to prefer to spend more time near the stimulus moving with acceleration when the higher mean velocity was used (Figure 14B, middle graph, p=0.4316). In all previous experiments, the period of habituation inside the central area of the tank was 10 minutes. Since this long period could decrease the interest of the focal fish in the stimuli, the time in the central area was reduced to 1 minute. This period combined with a 2.8 cm/s mean velocity elicited a significant preference for the animated dot, measured by the time spent in the region near this stimulus (Figure 14B, right graph, p<0.5). It was also tested if the preference towards the animated dot would vary with the trajectory of the dot. Dots moving with a single trajectory (the dot entered the screen from one side and left the screen through the opposite side) and with other less predictable trajectories were tested, namely a dot moving back and forward and a dot moving with multiple trajectories (Figure S3). When exposed to these different trajectories fish always spent more time near the animated dot, however this preference was not significant for more complex trajectories (Figure 14B, p= for the back and forward stimulus; p=0.25 for the stimulus with multiple trajectories). Therefore, in subsequent experiments the stimulus used was the one in which the dot moved with a single trajectory. All results presented bellow correspond only to the first 2 minutes of the 20 min trial, since only during this period a significant preference towards the animated dot was observed (Figure S4, Supplementary Material). 24

44 Figure 14. Adult zebrafish ability to detect acceleration cues (speed-changes) in a single object. A Effect of the dot size (0.8 versus 1.6 cm diameter) on the preference score towards the animated stimulus. The preference score represents the ratio of time spent by the subject in the region of interest near the animated stimulus (dot moving with acceleration) with respect to the time spent in both outer areas near a stimulus (dot with acceleration and dot with a constant speed). Data is presented as mean ± SEM, ns non-significant, between preference scores, Mann-Whitney test. B - Percentage cumulative time spent near the stimulus (animated versus inanimate dot) during the first 2 minutes of recording. Comparison between different mean velocities and different periods of habituation in the central area of the tank. C - Percentage cumulative time spent near the stimulus during the first 2 minutes of recording. Comparison between different trajectories (single, back and forward, multiple). Data is presented as mean ± SEM, ns non-significant, * p<0.05, Wilcoxon test. 25

45 5. CHAPTER 3- INVOLVEMENT OF OXYTOCIN-LIKE PEPTIDES IN MODULATING ADULT ZEBRAFISH RESPONSE TOWARDS ACCELERATION CUES As mentioned before, oxytocin-like peptides are involved in the regulation of several social behaviours across species. Therefore, with the purpose of determining if IT can modulate the ability to perceive animacy through acceleration cues, the behavioural paradigm previously established was tested using two different lines: a transgenic line in which there is an impairment in the vesicular release of IT and a mutant line where the IT receptor is truncated MATERIAL AND METHODS Animal housing and genetic screening All subjects used in this experiment (n=64) were four to eight month old male and female zebrafish from the transgenic line OXT:GAL4/UAS:BotxBLC and from the mutant line for the OXT receptor, bred and held at Instituto Gulbenkian de Ciência. Animals were kept in a water recirculation system at 28ºC in a 14h/light: 10h/dark cycle and the water quality was monitored every day: nitrites <0.2ppm, nitrates <50ppm, ammonia ppm, ph = 7 and conductivity at approximately 700 μsm. OXT:GAL4/UAS:BotxBLC The GAL4/UAS system is a technique used to generate transgenic lines under specific transcriptional control. It is composed by two elements derived from yeast, the GAL4 transcription factor, and its target sequence, the UAS (upstream activator sequence) element. By generating a transgenic line where the GAL4 factor is under the control of a specific promoter (in this case, IT) it is ensured that only the cells that express IT can transcribe the genes placed under the UAS element. The GAL4 was placed under the control of the IT gene promoter, and the UAS was placed upstream of the Botoxulin (Botx) gene that was tagged with a green fluorescent protein (GFP). With this system, only IT neurons will express the botoxulin protein which impairs vesicular release, thus affecting secretion in the isotocinergic system. In order to separate the three genotypes, a genetic screening was performed using a stereomicroscope (Leica MZ10 F). Larvae with 4 days post-fertilization were anaesthetized with Tricaine (MS-222) since at this stage it is possible to distinguish the three groups due to specific phenotypic differences: in the OXT:GAL4/UAS:BotxBLC (+,+) fish is possible to observe the IT neurons green as a result of the GFP tagging (Figure 15); the OXT:GAL4 larvae can be identified because it has a green marker for the heart, allowing it to be discriminated from the wild types or UAS:BOTxBLC fish, which are not labeled at all. These two last groups were used as controls for all the experiments with this transgenic line. 26

46 Figure 15. Left panel: Dorsal view of zebrafish larvae with isotocin neurons expressing BotxBLC-GFP. Right panel: Close up view of the isotocinergic neurons. Knockout line for the isotocin receptor This KO line was generated using a TALEN-Based Genome Editing system (Weizmann Institute) and is characterized by a single nucleotide deletion leading to a truncated IT receptor. Before the beginning of the behavioural test, the individuals were genotyped to separate the different genotypes: heterozygotes (+,-), wild type (+,+) and KO (-,-) fish. Subjects with three month old were anesthetized with 1x Tricaine (MS-222) in order to clip the tip of the caudal fin for genomic DNA extraction genomic. These samples were then collected in a microcentrifuge tube containing 50 μl of NaOH 50 mm and incubated at 95ºC for 20 minutes After the incubation the samples were cooled down before adding 1/10th of the volume of Tris-HCl (1 M, ph 8.0). The PCR reaction was performed using specific primers designed around the OXT-like receptor deletion site. After the PCR product was loaded in a 1% agarose gel, the agarose bands were cut and the DNA was purified using a commercial kit (NucleoSpinGel and PCR Clean-Up (Macherey-Nagel) and sent for sequencing (Figure 16). 27

47 Figure 16. Sequencing results - chromatogram for each of the three possible genotypes for the IT receptor KO line: wild type (+,+), heterozygotes (+,-),and KO (-,-) fish Behavioural paradigm The same protocol was used in this experiment with slight changes for optimization of the procedure, namely: (i) after the period of habituation, instead of exposing the focal fish to the stimuli for 10 minutes inside the central compartment, they were only exposed for 1 minute before the partitions were lifted and the subject was able to explore the entire arena; (ii) the exploration period was 6 minutes rather than 20 minutes, since it was observed that after this time-point the fish s response became inconsistent since the focal fish was less attentive to the stimuli. One region of interest (ROI) was defined near each screen and the preference towards a stimulus was measured by the time spent in this region (Equation 1.1.) Video analysis All behavioural tests were recorded using fixed infra-red cameras at an acquisition rate of 30 fps, connected to a laptop using the video recording software, Pinnacle Studio 12 ( The analysis of the behaviour was performed using the videotracking software Ethovision XT11 from Noldus ( 28

48 Statistical analysis Normality was verified using D'Agostino & Pearson normality test. The data from the present experiment were analyzed with a one-way ANOVA, except the preference score by sex that was analyzed with a two-way ANOVA. In the Supplementary Material, the locomotor activity for both groups was analyzed with an unpaired t test. Outliers analysis was performed using ROUT test (Q = 1%). Descriptive statistics were presented as mean ± SEM. Statistical significance was set at p < 0.05 (*), p < 0.01 (**), p < (***) and p < (****). All statistical analyses were performed with GraphPad Prism 6 ( 29

49 5.2. RESULTS To test if the ability to perceive animacy can be modulated by IT, the previous behavioural task was performed using two different lines: a transgenic line in which there is an impairment in the vesicular release of IT and a mutant line for the IT receptor (see Methods). Contrary to what was expected, the ability to discriminate between an animated and an inanimate stimulus was not lost in the transgenic fish. In fact, in the male transgenic fish this ability to discriminate is present not only in the first 2 minutes of the trial, but during the whole 6-minute trial (Figure 17A). More than discriminating both stimuli, male transgenic fish seem to prefer the animated stimulus over the inanimate stimulus, translated by the fact that they spent the entire trial near the first one and also by the high preference score towards acceleration (Figure 18). On the other hand, although there was a tendency for male wild type fish to spend more time near the animated stimulus, this tendency is not significant and the preference score is relatively low (Figure 17A; Figure 18). This finding contradicts what was previously described when optimizing the protocol and could, therefore, be explained by a variability related to the line itself. Regarding the female fish, both the control and the transgenic group are able to discriminate between the two stimuli and spent more time near the dot moving with acceleration when compared to the inanimate dot, not only during the first 2 min of the trial, but also during the entire trial (Figure 17B). Despite this pattern, female transgenic fish still displayed a higher preference score for acceleration (Figure 18). As regards the preference score towards acceleration during the first 2 minutes of the trial, there is an effect of the genotype; both male and female transgenic fish present a higher score when compared to wild type fish. We can also observe a significant effect of sex, with females in both groups presenting a higher score when compared to males (Figure 18; Table 1). When comparing the locomotor activity (translated by the total distance covered and the mean velocity) of the wild type and the transgenic fish, we can observe no significant differences indicating that the different behavioural responses are not due to anxiety-like behaviours (Figure S5, Supplementary Material). 30

50 Figure 17. Involvement of isotocin in modulating adult zebrafish response towards acceleration cues in a transgenic line with impaired isotocin vesicular release. A Percentage cumulative time spent near the stimulus (animated versus inanimate dot) during a 6 min. trial (left panel) and the first 2 min. of the trial (right panel), in male adult zebrafish. B - Percentage cumulative time spent near the stimulus (animated versus inanimate dot) during a 6 min. trial (left panel) and the first 2 min. of the trial (right panel), in female adult zebrafish. Data is presented as mean ± SEM, ns non-significant, *** p<0.001, **** p< Ordinary one-way ANOVA. 31

51 Figure 18. Involvement of isotocin in modulating adult zebrafish preference towards acceleration cues in a transgenic line with impaired isotocin vesicular release. Comparison between the preference score towards the animated dot, regarding the sex and the genotype. The preference score represents the ratio of time spent by the subject in the region of interest near the animated stimulus (dot moving with acceleration) with respect to the time spent in both outer areas near a stimulus (dot with acceleration and dot with a constant speed). Data is presented as mean ± SEM. Significant effect of sex and genotype. Two-way ANOVA. Table 1. Effect of sex and genotype (WT or OXT, UAS:BOTxBLC) on the preference score towards the animated stimulus. Significant effect of sex (F(1,55), p<0.05) and genotype (F(1,55), p<0.01). No significant interaction between sex and genotype. Two-way ANOVA. Effects F (DFn, DFd) P value Sex F (1, 55) = Genotype F (1, 55) = Interaction F (1, 55) =

52 Concerning the mutant line, slightly differences were observed compared to the transgenic line. Male fish in both groups are able to discriminate between an animated and an inanimate stimulus and preferred to spend more time near the animated stimulus. However, for the WT animals this pattern was more evident during the first 2 minutes of the trial, suggesting that these fish explored more the entire arena during the rest of the trial. The mutant male fish spent almost the whole trial near the animated stimulus, barely exploring the remaining arena (Figure 19A). The behavioural response observed in female fish is relatively similar: both groups discriminate between the two stimuli and spent more time in the region near the animated stimulus. In both wild type and mutant female fish there were no differences between the behavioural response in the first 2 minutes of the trial and in the whole 6-minute trial, indicating that females spent the entire trial near the dot that moved with acceleration (Figure 19B). Concerning the preference score towards acceleration in the first 2 minutes of the trial, there was no effect of the line, although mutant male fish presented a higher score when compared to WT fish. On the other hand, there was an effect of the sex, with male mutant fish presenting a higher score than female mutant fish, which can be explained by the low sample size used for the females in this experiment (Figure 20). No significant differences were registered regarding the locomotor activity (Figure S6, Supplementary Material). 33

53 Figure 19. Involvement of isotocin in modulating adult zebrafish response towards acceleration cues in an isotocin receptor mutant line. A Percentage cumulative time spent near the stimulus (animated versus inanimate dot) during a 6 min. trial (left panel) and the first 2 min. of the trial (right panel), in male adult zebrafish. B - Percentage cumulative time spent near the stimulus (animated versus inanimate dot) during a 6 min. trial (left panel) and the first 2 min. of the trial (right panel), in female adult zebrafish. Data is presented as mean ± SEM, * p<0.05, ** p<0.01, *** p<0.001, **** p< Ordinary one-way ANOVA. 34

54 Figure 20. Involvement of isotocin in modulating adult zebrafish preference towards acceleration cues in an OXTr mutant line. Comparison between the preference score towards the animated dot, regarding the sex and the genotype. The preference score represents the ratio of time spent by the subject in the region of interest near the animated stimulus (dot moving with acceleration) with respect to the time spent in both outer areas near a stimulus (dot with acceleration and dot with a constant speed). Data is presented as mean ± SEM. Effect of sex but no effect of genotype. Two-way ANOVA. Table 2. Effect of sex and genotype (WT or OXTr KO) on the preference score towards the animated stimulus. Significant effect of sex (F(1,55), p<0.001). No significant effect of the genotype and no significant interaction between sex and genotype. Two-way ANOVA. Effects F (DFn, DFd) P value Sex F (1, 39) = Genotype F (1, 39) = Interaction F (1, 39) =

55 6. CHAPTER 4: CHARACTERIZATION OF THE NEURAL CIRCUITRY UNDERLYING ANIMACY PERCEPTION (C-FOS ACTIVITY) 6.1. MATERIAL AND METHODS Animal housing All subjects used in this experiment (n=4) were five month old male zebrafish from an outcross wild-type TL strain bred and held at Instituto Gulbenkian de Ciência Animals were kept in a water recirculation system,at 28ºC in a 14h/light: 10h/dark cycle and the water quality was monitored every day: nitrites <0.2ppm, nitrates <50ppm, ammonia ppm, ph = 7 and conductivity at approximately 700 μsm Behavioural paradigm For this task, it was used the behavioural setup demonstrated in Figure 7 with one opaque partition and one transparent partition, instead of two transparent partitions as previously described. With this modification to the setup, each fish was only able to visualize one of the three groups of stimuli: i) video of an empty tank (control group); ii) video of an inanimate entity (dot moving with a constant speed); iii) video of an animated entity (dot moving with an acceleration during its trajectory). The focal fish was placed in the central compartment and exposed to a video of an empty tank during 10 minutes for acclimatization; after this period one of the three stimuli appeared in one of the screens for 1 minute before the transparent partition was lifted and the fish was allowed to explore the stimulus for 2 more minutes. In this protocol the exploration period was shorter than what was described in the previous experiments in order to ensure that the focal individual did not lose the interest for the stimulus. The experiment was video-recorded for future video-tracking. In parallel and as a positive control for both techniques, fish were exposed to PTZ (Pentylenetetrazole), a central nervous system convulsant Sampling For in situ hybridization, the focal fish was sacrificed with and overdose of tricaine (MS- 222, 25X) 30 minutes after the first exposure to the stimulus. For immunohistochemistry, the focal fish was sacrificed 90 minutes after this first exposure. Since the individuals were in the presence of the stimulus only for 3 minutes (1 minute inside the partitions and 2 minutes to explore the stimulus), they remained in the experimental tank for more 27 or 87 minutes to reach the peak of c-fos mrna expression or c-fos protein, respectively. During this period, the fish were only exposed to a video of an empty tank. After sacrificing the fish, heads were collected and fixated with 4% paraformaldehyde (PFA) at 4ºC overnight. On the subsequent day, brains were extracted and placed in 4% PFA during 1 hour for post-fixation and then were cryoprotected using a 34% sucrose solution. Finally, brains were embedded in OCT (Tissue-Tek ) and were cryosected in 16 µm slices that were stored at -20ºC. 36

56 In situ hybridization Obtaining plasmidic DNA and probe synthesis Bacteria (Escherichia coli) containing both the plasmid of interest and a resistance to the antibiotic kanamycin were grown in a LB medium containing kanamycin (direct inoculation). Plasmidic DNA was then extracted using a Plasmid Miniprep kit (Zyppy ). After obtaining the plasmidic DNA the plasmid was linearized, starting by preparing a linearization mix that was then incubated overnight at 37ºC: 15 µl of plasmidic DNA, 10 µl of restriction enzyme buffer (10X), 3 µl of restriction enzyme (BAM HI), 0.5 µl of BSA (10mg/ml) and DEPC treated water up to 100 µl. Next day, after confirming through a 1% agarose gel that all the plasmid was digested, DNA was purified by extraction with PCIA. After adding the PCIA (200 µl of PCIA for 200 µl of solution), the mixture was centrifuged for 5 minutes, 1400 rpm at room temperature to separate the 2 phases. The aqueous upper phase was transferred a fresh tube and after adding 3M Sodium Acetate (ph 5.2), 400 µl of absolute ethanol were added and the mixture was precipitated at -20ºC overnight. On the following day, after a centrifugation of 30 minutes, 1400 rpm at 4ºC, the supernatant was discarded and the mixture was washed with 1ml of 70% ethanol and centrifuged for 5 minutes, 1400 rpm at room temperature. The ethanol was removed and the pellet was left to dry on the bench before preparing the transcription reaction mix: 2 µl of linear DNA, 2 µl of transcription buffer (10X), 2 µl of DIG labelling mix (10X), 2 µl RNA polymerase (T7 for the antisense probe), 1 µl of RNase inhibitor and DEPC treated water up to 20 µl. After a spin-down the mixture was incubated at 37ºC for 2h. After this incubation period, 1 µl of RNase-free DNase I was added and the mix was incubated for more 15 minutes. An aliquot was checked in a 1% agarose gel to assess if this incubation removed the remaining DNA efficiently. The sample was then precipitated by adding: 180 µl of DEPC-treated water, 22 µl 3M Sodium Acetate (ph 5.2) and 500 µl of absolute ethanol and stored at -20ºC overnight. Next, the pellet was precipitated in microfuge for 30 minutes, 1400 rpm at 4ºC and washed with 70% ethanol for 5 minutes. Finally, the pellet was resuspended in 30 µl DEPCtreated water and, after checking if the probe can be seen in a 1% agarose gel, 30 µl of deionized formamide were added. The probe was stored at -20ºC until required for in situ hybridization In situ hybridization in slices The slides were transferred from -20ºC to room temperature for 15 minutes and were post-fixated with 4% PFA for 10 minutes at room temperature. Then, the slides were washed with PBS (3x5 minutes) and treated for 10 minutes with an acetylation mix: 1ml of DEPCtreated water per slide with 11.2 µl triethanolamine and 2.5 µl acetic anhydride. After washing again with PBS (3x5 minutes), the slides were prehybridized horizontally with enough volume of prehybridization solution at 68ºC in a humidified chamber for approximately 5 hours. Next, 200 µl of probe were diluted in prehybridization solution and the slides were hybridized horizontally in a humidified box at 68ºC overnight. Slides were coverslipped to prevent probe s evaporation. 37

57 The slides were washed with a pre-warmed solution 1 containing 50ml 50% formamide, 25ml 5X SSC ph 4.5, 10ml 1% SDS and DEPC-treated water up to 100ml, for 5 minutes at 68ºC. After this quick wash, another one was performed for 1 hour at 68ºC. Next, the slides were washed with a pre-warmed solution 2 containing 50ml 25% formamide, 5ml 2X SSC ph 4.5, 0.5ml 1% Tween20 and DEPC-treated water up to 50ml, for 1 hour at 68ºC. After washing 2x with TBST for 5-10 minutes, the slides were transferred to a tray and were blocked with blocking buffer (10% HINGS in TBST) for 40 minutes at room temperature. Further, the slides were incubated in blocking buffer with anti-dig antibody (1:2000) for 80 minutes at room temperature and then were washed overnight at 4ºC with TBST to reduce background. Slides were washed in NTMT 4x for 5 minutes. This solution was prepared as follows: 1ml 5M NaCl, 2.5 ml 1M MgCl 2, 2,5ml 2M Tris ph 9.5, 500 µl Tween20 and DEPC-treated water up to 50 ml; and was then filtered through a 0.45 µm filter. Slides were incubated in a humidified chamber with a staining solution (4.5 µl NBT and 3.5 µl BCIP per 1ml of NTMT) that was previously filtered (0.2 µm filter), until staining is complete. The reaction was stopped with PBS washes and the slides were mounted with glycergel (DAKO ) Immunohistochemistry The slides were transferred from -20ºC to an oven at 37ºC for 30 minutes. After washing with TBS 3X 5 minutes each, it is necessary to unmask the epitope incubating the slides in Tris-EDTA (10mM Tris, 1mM EDTA) and 0.05% Tween20 ph 9.0 for 20ºC at 95ºC. Next, the slides were left to cool-down at room temperature for 5 minutes. Thereafter, slides were washed with TBS 0,025% Triton X-100 2X 5 minutes each and then were blocked with TBS with 1% BSA for 1 hour at room temperature, to avoid unspecific binding of the secondary antibody. Following this period, slides were incubated with the primary antibody rabbit anticfos (1:200 in TBS + 1% BSA) in a humidified chamber at 4ºC overnight. On the following day, slides were washed with TBS 0,025% Triton X-100 2X 10 minutes each and were incubated with the secondary antibody goat anti-rabbit (1:300 in TBS + 1% BSA) for 2 hours at room temperature. After this incubation period, slides were again washed with TBS 0,025% Triton X-100 3X 5 minutes each. Finally, slides were treated with DAPI (1:1000 in TBS) and washed with TBS 0,025% Triton X-100 2X 5 minutes each before they were mounted with a fluorescence mounting medium from DAKO. 38

58 6.2. RESULTS In order to determine which brain region are activated during animacy perception through acceleration cues, we assessed the expression of a marker for neuronal activation, c-fos and its product c-fos, resorting to in situ hybridization or immunohistochemistry, respectively. We present in this section a demonstrative example for each of the techniques described above (n = 2), focusing on the section 85 from the zebrafish brain atlas (Figure 21) since this section includes regions that are included in the Social-Decision Making Network (O Conell et al., 2012). Here we highlight three specific regions: Dm (medial zone of the dorsal telencephalic area), Vd (dorsal nucleus of the ventral telencephalic area) and Vv (ventral nucleus of the ventral telencephalic area). Since these experiment are still ongoing, it is not possible to observe if there are in fact differences between the brain regions activated when an individual is exposed to an animated (acceleration) or an inanimate dot (no acceleration). In fact, regarding the in situ hybridization assay, the example presented demonstrates a similar activation of the selected regions between the two groups (Figure 22) Therefore, it is necessary to increase this sample size and to compare the number of c-fos positive cells in fish exposed to these different stimuli. Figure 21. In situ hybridization of c-fos reveals neural activity when fish are exposed to specific visual stimuli. A c- fos expression in the left brain hemisphere (and the correspondent right brain hemisphere from the section 85 from the zebrafish brain atlas) of an adult zebrafish exposed to an inanimate stimulus (dot moving with constant speed); activation of Dm, Vd and VV; 4X magnification. B - c-fos expression in the left brain hemisphere (and the correspondent right brain hemisphere from the section 85 from the zebrafish brain atlas) of an adult zebrafish exposed to an animate stimulus (dot moving with acceleration); activation of Dm, Vd and Vv; 4X magnification. C- section through the telencephalic region, corresponding to section 85 in the zebrafish brain atlas (Wulliman et al., 1996). Highlight for the regions represented in 21A and 21B. Dm medial zone of the dorsal telencephalic area; Vd dorsal nucleus of the ventral telencephalic area; Vv ventral nucleus of the ventral telencephalic area. 39

59 Figure 22. Mapping of c-fos neural activity. c-fos positive cells are shown in dark blue Left Brain section of a focal fish that was exposed to an inanimate stimulus (dot moving with constant speed); 20X magnification. Right - Brain section of a focal fish that was exposed an animate stimulus (dot moving with acceleration); 20X magnification. A - Dm (medial zone of the dorsal telencephalic area). B - Vd (dorsal nucleus of the ventral telencephalic area). C - Vv (ventral nucleus of the ventral telencephalic area). When conducting in situ hybridization it is essential to test also a positive control in order to confirm that the technique is in fact working properly. Therefore, we present here a previously conducted experiment, in which an adult zebrafish was exposed to PTZ, a convulsant that should elicit high levels of c-fos activation across different brain regions (Figure 23). In this assay the amount of background is lower than the one we can observe in Figure 22, which probably indicates that the in situ hybridization assay or even the behavioural protocol used for this experiment still need further optimization. 40

60 Figure 23. In situ hybridization of c-fos reveals neural activity when fish are exposed to PTZ (positive control). Left Brain section of a focal fish that was exposed to a PTZ treatment; 10X magnification. Right - Brain section of a control focal fish that was only exposed to water; 10X magnification. Concerning the immunohistochemistry assay, it seems that the exposure to an animated stimulus elicited a higher number of c-fos immunoreactive-positive cells, for both Dm and Vv (Figure 24; Figure 25). However, these results represent one single fish per group and so it is not possible to make any conclusions. Furthermore, it is important to notice that the signal for the experimental fish is not as clear as the signal that is observed in the positive control with PTZ. 41

61 Figure 24. Immunohistochemistry for c-fos activity in Dm (medial zone of the dorsal telencephalic area). Left - c-fos immunoreactive-positive cells; Center - DAPI immunoreactive-positive cells; Right - counterstained brain sections with the nuclear fluorescent stain DAPI (blue) merged with c-fos immunoreactive-positive cells (blue). Each line represents a different treatment: PTZ treatment (top panel); exposure to an animated stimulus (middle panel); exposure to an inanimate stimulus (lower panel). 42

62 Figure 25. Immunohistochemistry for c-fos activity in Vv (ventral nucleus of the ventral telencephalic area). Left - c-fos immunoreactive-positive cells; Center - DAPI immunoreactive-positive cells; Right - counterstained brain sections with the nuclear fluorescent stain DAPI (blue) merged with c-fos immunoreactive-positive cells (blue). Each line represents a different treatment: PTZ treatment (top panel); exposure to an animated stimulus (middle panel); exposure to an inanimate stimulus (lower panel). 43

63 7. DISCUSSION In the present work we demonstrated, to the best of our knowledge for the first time, that adult zebrafish are able to perceive animacy, through acceleration cues. Moreover, we have shown that there are behavioural differences in males and females regarding the response towards animacy cues. Furthermore, as we predicted, IT seems to modulate this social-cognitive ability. In this project we started by conducting a preliminary experiment with the purpose of corroborating that zebrafish, as a highly social species (Oliveira et al., 2013; Spence et al., 2008) are more motivated to approach a social stimulus, rather than a non-social stimulus. As expected, it was observed that zebrafish spent more time in the region of interest near the stimulus when exposed to a conspecific (social stimulus) than when exposed to an object (nonsocial stimulus) or an empty tank (control group). In these latter exposures, the fish spent more time exploring the entire arena. The fish that were exposed to a conspecific presented decreased locomotor activity, which was expected since this is an indicator of anxiety and the possibility to shoal with a conspecific decreases the anxiety levels in this species (Kalueff et al., 2013). In this experiment fish were isolated for 8 days in order to increase the response towards a social stimulus. However, this period could be considered too extensive for a species that usually lives in a group, inducing high levels of stress. Thus, we conducted a supplementary experiment to assess if fish isolated overnight would behave differently from the fish of our previous experiment that were isolated for 8 days before the test. When fish was exposed to a conspecific, no differences were found in the time spent in ROI and in the locomotor activity between fish isolated for 8 days or isolated overnight. On the other hand, individuals that were exposed to an object spent more time in the opposite side of the stimulus, rather than exploring the whole arena equally as it happened in the control group. Therefore, it was hypothesized that these subjects were in avoidance with the object, probably due to the fact that they had been isolated for 8 days before this exposure. So, it would be important to confirm this hypothesis with a supplementary experiment, similar to the one we described before. Nevertheless, observing the results presented in the Supplementary Material for the optimization of the object can give us some insights regarding this period of isolation, since in this experiment fish were only isolated overnight and explored the entire arena evenly when exposed to an object. Although the results obtained in this experiment are in accordance to what was expected, it is necessary to take into consideration that one stimulus is moving (conspecific) and the other is static (object) and this fact could by itself explain why fish are more motivated to approach the social stimulus when compared to the non-social stimulus. For this reason, it would be relevant to test if zebrafish would spend more time near a non-stimulus if this one was moving, using for example video playbacks to present these stimuli. Nevertheless, this experiment along with other results from the literature (Saverion et al., 2008; Spence et al., 2008; Engeszer et al., 2007; Miller et al., 2011) show that zebrafish prefer to associate with other conspecifics, which can be due to specific features that characterize the motion of a living organism. 44

64 Therefore, in order to test this hypothesis, it was essential to assess if zebrafish is able to detect animacy cues and to determine if they prefer in fact to spend more time in association with an animated agent, as it was already shown in other species (Tremoulet et al., 2000; Kovács et al., 2016; Mascalzoni et al., 2010; Goto et al., 2000; Tsutsumi et al., 2012; Nakayasu et al., 2014; MacKinnon et al., 2010). In all the subsequent experiments we used video playbacks as a tool to present the stimuli. Video playbacks are a powerful tool to study visual communication in animals, and have been widely used in fish in different social paradigms such as social preference and matechoice (Saverino et al., 2008; Qin et al., 2014). Moreover, this tool allows more controllable and repeatable visual stimuli than the use of live animals and is highly suitable for studying motion, among other features (i.e. shape, size). However, since all video systems are designed for the human vision, it was crucial to determine if zebrafish could perceive stimuli that were presented using this system. So, to validate the use of video playbacks in this species, we took advantage of the fact that zebrafish prefer to associate with conspecifics. As predicted, when exposed to a video of a mixed shoal in one side of the setup and a video of an empty tank on the opposite side, individuals spent the whole trial in the region of interest near the shoal, barely exploring the remaining arena. The same pattern is observed when the fish had to choose between spending more time near a video of a single conspecific or the same video of an empty tank. Although the majority of the shoal preference protocols use a real shoal opposed to an empty tank (Wright et al., 2006), we intended to confirm that fish were not approaching the video of the shoal or the video of the conspecific simply because they presented motion, contrary to the video of an empty tank. Therefore, individuals were exposed at the same time to two stimuli with motion: a video of a shoal in opposition to a video of a conspecific. Similarly to what is observed with real fish, zebrafish prefer to spend more time near the shoal rather than the single fish. In this experiment, instead of using a mixed shoal we used a shoal of males when the stimulus conspecific was a male and a shoal of females when it was a female stimulus conspecific. We performed this combination in order to avoid that the focal male would avoid the shoal stimulus to engage in a fight with the male conspecific stimulus or display a mating behaviour with the female conspecific stimulus (Fernandes et al., 2015). Thus, by using the same sex for the shoal that was being presented in opposition to the single fish, we ensured that the focal individual could exhibit the previously mentioned behaviours both with the shoal and the conspecific stimuli. Concerning the validation of video playbacks as a tool to present stimuli, it would be relevant to compare zebrafish s behaviour when in the presence of a real fish or a video of a fish. This analysis was not performed in this project since the behavioural setups and the experimental protocols used for both experiments were highly distinct from each other. As previously mentioned, even simple geometric shapes can elicit high-level percepts of animacy if they display changes in speed and/or changes in direction or if they appear to be self-propelled agents. In order to disentangle which specific motion features that mediate animacy perception in zebrafish, it is necessary to evaluate this species response towards each one of them separately. 45

65 In fact, several studies have already shown that both humans and other species (e.g. chickens) interpret agents that have a self-propelled motion (Cicchino et al., 2011; Pratt et al., 2010; Di Giorgio et al., 2016) or that accelerate during their trajectory (Frankenhuis et al., 2013; Salva et al., 2016) as animated agents, in other words, as living entities, preferring to approach and associate with this agents compared to the inanimate ones. In this project we focused in one particular animacy cue: speed changes (i.e. acceleration), displayed by a black dot. First, when generating this type of stimuli it is fundamental to consider features such as its size and mean velocity. Although we did not find any significant differences in the preference towards the animated agent when using two different dot sizes, we believe that using a dot with the medium area of an adult zebrafish is the most indicated approach. This way, we avoid using a smaller dot that could elicit a prey capture response or a larger dot that could be interpreted as a predator and induce an escape response (Semmelhack et al., 2014; Dunn et al., 2017). On the other hand, it should have been used a dot whose mean velocity was comparable to zebrafish s mean velocity. However, since the distance covered by each dot was only 15 cm (corresponding to the width of the tank) this mean velocity was not possible to achieve, otherwise it could be difficult for the individuals to perceive what is being presented, considering that the dot would be entering and exiting the screen in a considerably short period of time. Despite the fact that the two mean velocities tested were not similar to the real speed of an adult zebrafish ( 7 cm/s), we observed that the highest mean velocity of the two that were tested was sufficient to elicit a tendency to spend more time near the animated stimulus. Nevertheless, this tendency only became significant when the time spent habituating to these stimuli inside the partitions was reduced from 10 to 1 minute, which can be expected since the long period of habituation could diminish focal fish s interest towards the stimuli when they were choosing between the animated and the inanimate agent. In this experiment, we used a simple trajectory in which the dot was entering the screen in one side and leaving the screen through the other. However, in our stimuli-videos, the dot was always entering by the left side and leaving through the right side. Therefore, it was important to evaluate if using more unpredictable trajectories would elicit a different response in the focal fish. Although zebrafish showed a tendency to spend more time near the animated agent with these new stimuli, this tendency was not significant probably because these stimuli were more complex and, therefore, more difficult to interpret. After the stimuli optimization mentioned above, it was possible to verify that zebrafish can in fact perceive animacy through acceleration cues and prefer to associate with an animated agent rather than an inanimate agent. Nevertheless, with these stimuli it is not possible to determine if zebrafish s attention is driven by the precise moment in which the dot starts to accelerate or by the moment in which the dot is moving at a higher speed after the onset of the acceleration (Abrams et al., 2003). Therefore, it would be interesting to add another experiment to disentangle this question by adding a stimulus in which the onset of the acceleration is hidden by occluders (bars) and individuals can only visualize the dot moving with a higher speed when compared to the beginning of its trajectory. If with this stimulus zebrafish loose the preference towards the animated agent it means that the particular moment in which the dot starts accelerating is crucial to perceive animacy, as it was shown by Rosa-Salva et al (Salva et al., 2016). 46

66 Regarding the stimuli we used in this experiment, it is also important to notice that both the animated and the inanimate stimuli entered the screen already in motion, which means that both stimuli were ambiguous since the focal fish could not assess if they were selfpropelled or not. So, it would be important to test other stimuli in which the subjects could see the animated agent start moving from a resting position without any external energy source applied to it and determine if using this stimulus would elicit a higher level percept of animacy. In fact, we implemented a similar experiment but the zebrafish did not display a preference towards the self-propelled stimulus compared to the ambiguous stimulus (Data not shown). Therefore, further studies are needed to unravel this question. Moreover, in future experiments we are planning to assess if other features of animacy, such as changes in direction, can elicit a preference for the animated dot. If so, it would be relevant to understand if having an agent moving with both accelerations and changes in direction during its trajectory would trigger a higher preference towards the animated dot when compared to stimuli moving only with accelerations or changes in direction. Oxytocin and oxytocin-like peptides are known to modulate a myriad of social behaviours across taxa (Goodson et al., 2010; Goodson et al., 2013). In fact, it has already been demonstrated that oxytocin seems to influence biological motion perception not only in humans but also in other species (e.g. dogs) (Kéri et al., 2009; Kovács et al., 2016). Nonetheless, it has never been shown if these peptides modulate the perception of simple animacy cues. Therefore, one of the goals of this project was to determine if IT (the oxytocin homologue in teleost fish) regulates animacy perception, more specifically, acceleration cues. In order to achieve this goal, we tested the previously optimized protocol in a transgenic and a mutant line whose isotocinergic system is impaired (see Methods). We were expecting that an impairment in the isotocinergic system would affect the ability to perceive animacy cues. However, contrary to what was predicted, neither the transgenic nor the mutant fish lost the preference towards the animated stimulus. In fact, we observed that while male wild type individuals explored the animated stimulus especially in the first 2 minutes of the trial, male transgenic and mutant fish spent not only the first 2 minutes but the whole 6-minute trial near the animated stimulus. This difference could be explained by the existence of attention deficits associated with this impairment in the isotocinergic system. We could hypothesize that transgenic and mutant fish are not able to easily perceive the animacy stimulus, therefore they seem to be fixed in the stimulus in order to retrieve the information that wild type fish are capable of retrieving apparently in the first 2 minutes of the trial (Kovács et al., 2016). Thus, it would be relevant to test these lines in behavioural paradigms that evaluate attention abilities, especially considering that oxytocin has already been associated to attention deficits in humans (Sasaki et al., 2015; Demirci et al., 2016). Interestingly, this pattern was not observed in female fish. Female fish, both wild type and transgenic/mutant, displayed a significant preference for the animated stimulus during the entire trial, not only for the first 2 minutes. Based on these results, it seems that male and female wild type fish perceive animacy differently: while male fish probably retrieve the information from the animated stimulus in 47

67 the first 2 minutes and then explore the other stimulus and the remaining arena, females spend the majority of the trial near the animated stimulus. Furthermore, the data obtained for the transgenic and the mutant lines suggests that IT only modulates the perception of animacy cues in male fish. These outcomes could be justified by a different distribution of IT receptors, which has already been pointed out by other studies (Banerjee et al., 2017; Duchemin et al., 2017; Dumais et al., 2016). Nevertheless, it is important to mention that the number of fish tested for the female IT receptor mutant group is lower than the number of fish tested for the other groups, so we cannot be certain that we would obtain the same results if we increased this sample size in this group. In fact, in the transgenic line, males never display a significant preference towards the animated stimulus one compared to the inanimate one, although there is a tendency to spend more time near the first stimulus. So, it would also be important to increase the sample size for this line. All the aforementioned results point out the necessity to look to the brain regions activated during animacy perception in order to disentangle the neural mechanisms underlying this social-cognitive ability. Furthermore, these results might help us to explain the differences obtained between wild type and transgenic/mutant fish and also between male and female wild type fish. As previously stated, the activation of these brain regions can be assessed through in situ hybridization or immunohistochemistry assays for immediate-early genes, such as, c-fos. It is important to conduct both assays since they measure different products (messenger RNA and protein, respectively) and, therefore, with these two techniques we might identify different activated brain regions (Kovács et al., 2008; Kovács et al., 1998). In fact, these experiments are already ongoing, and although we cannot yet retrieve any conclusions, we present in the Results section a demonstrative example for each of the assays performed in male wild type fish. Besides wild type fish, transgenic and mutant fish are also being evaluated, considering both sexes. From the examples we show it is possible to notice both techniques still need to be further optimized. Regarding the in situ hybridization, we can detect a considerable amount of background, which could difficult the distinction of c-fos positive cells. On the other hand, in the immunohistochemistry we can verify a strong c-fos activation in the brain exposed to PTZ, but the same was not observed in fish exposed to either visual stimuli. As far as we know, there are not yet any studies that have determined which brain regions are activated during the perception of acceleration cues. However, we are expecting that this animated stimulus elicits the activation of brain regions contemplated in the "Social Behavior Network" described by Newman (1999), such as the ventral nucleus of ventral telencephalic area (Vv; lateral septum in mammals) that has been widely associated with social behaviour in a broader sense (Newman, 1999; Goodson et al., 2005; Sheehan et al., 2000). Naturally, other regions connected to visual processing, such as the optic tectum, are also expected to be active (Salva et al., 2015). Moreover, it would also be interesting to conduct an in situ hybridization assay for the IT receptor together with an immunohistochemistry for c-fos to determine if there is a colocalization and assess if there is still an activation of the isotocinergic pathway in transgenic and mutant fish that could explain the results obtained for these lines, focusing in brain regions that have been shown to present receptors for IT in social fish (Huffman et al., 2012). 48

68 8. CONCLUSIONS The present work demonstrated, for the first time, that zebrafish are able to perceive animacy through acceleration cues and, as a social species, these individuals are more motivated to approach this animated stimulus. However, the percentage of time spent near the animated dot was considerably lower compared do the time fish spent near the shoal or the conspecific presented in the video playbacks. This result was expected since we only use acceleration as animacy cue, which by itself cannot accurately represent the motion of a living organism. Therefore, as mentioned before, future studies should address other animacy cues, as changes in direction, in order to disentangle which specific motion features drive individuals' attention. Moreover, it is important to take into consideration the fact that the dot's shape is not similar to a zebrafish's shape and this aspect could also influence subjects' preference towards a stimulus (Saverino et al., 2008). Additionally, this study seems to illustrate differences between how males and females perceive animacy cues. In our experiments it was possible to observe that male zebrafish have a preference for the animated stimulus especially in the first 2 minutes of the trial, whereas females show this preference during the whole trial. Even when analyzing the results obtained for the transgenic and the ITr mutant line, we can identify differences between both sexes. Widtype females and transgenic females respond equally to the animated stimulus and the same pattern seems to be observed for the mutant line, despite the low sample size. However, males with an impairment in the isotocinergic system respond differently from the wild type males: instead of demonstrating a preference for the animated dot only in the first 2 minutes, this preference is significant during the whole 6-minute trial. Here we hypothesize that male and female zebrafish have a different distribution of IT receptors and that male fish with an impairment in the isotocinergic system might present attention deficits. All the aforementioned findings highlight the necessity of determining which brain regions are being activated during animacy perception, in order to disentangle the mechanisms underlying this social-cognitive ability that is essential for survival. 49

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77 10. SUPPLEMENTARY MATERIAL Figure S1. Effect of isolation on zebrafish behaviour. A Percentage cumulative time spent near the stimulus (conspecific) regarding two different periods of isolation: 8 days of isolation (dark grey bar) and overnight isolation (grey bar with stripes). Data is presented as mean ± SEM, ns non-significant, Mann- Whitney test. B Effect of social and non-social stimuli on the locomotor activity of the focal fish. Right panel Total distance covered by the focal fish during the whole trial regarding two different periods of isolation. Left panel Mean velocity of the focal fish during the entire trial in two distinct periods of isolation. Data is presented as mean ± SEM, ns non-significant, Mann-Whitney test. 58

78 Figure S2. Optimization of zebrafish s response to an object used as a non-social stimulus. Left panel Percentage cumulative time spent near the stimulus (object), comparing two distinct colours (red and green) and three different sizes: big (2ml Eppendorf tube), intermediate (1,5ml Eppendorf tube) and small (0,5 Eppendorf tube). B Percentage cumulative time spent in avoidance with the stimulus (meaning time spent in the opposite region to the one where the object was placed), comparing two distinct colours (red and green) and three different sizes: big (2ml Eppendorf tube), intermediate (1,5ml Eppendorf tube) and small (0,5 Eppendorf tube). Data is presented as mean ± SEM. 59

79 Figure S3. Schematic representation of the first 20 secs of the multiple trajectories stimulus. Upper panel: dot moving with a constant speed. Lower panel: dot in which there is a speed-change (acceleration) during its trajectory. 60

80 Figure S4. Percentage cumulative time spent in the stimulus (animated versus inanimate dot) during the entire trial, presented in 1 minute time bins. The area delimited by a red line represents the first 2 minutes of the trial in which the preference towards the dot moving with acceleration is significantly higher. Data is presented as mean ± SEM. 61

81 Figure S5. Involvement of isotocin in modulating adult zebrafish response towards acceleration cues in a transgenic line with impaired isotocin vesicular release. A - Upper panel: Comparison between the locomotor activity (Total distance and Mean Velocity) of male wildtype and male transgenic fish. Lower panel Comparison between the locomotor activity (Total distance and Mean Velocity) of female wildtype and female transgenic fish. Data is presented as mean ± SEM; ns non-significant, unpaired t test. 62

82 Figure S6. Involvement of isotocin in modulating adult zebrafish response towards acceleration cues in an oxytocin-receptor mutant line. A - Upper panel: Comparison between the locomotor activity (Total distance and Mean Velocity) of male wildtype and male mutant fish. Lower panel Comparison between the locomotor activity (Total distance and Mean Velocity) of female wildtype and female mutant fish. Data is presented as mean ± SEM; ns non-significant, unpaired t test. 63