Autism-associated gene shank3 is necessary for social contagion in zebrafish.

BACKGROUND: Animal models enable targeting autism-associated genes, such as the shank3 gene, to assess their impact on behavioural phenotypes. However, this is often limited to simple behaviours relevant for social interaction. Social contagion is a complex phenotype forming the basis of human empathic behaviour and involves attention to the behaviour of others for recognizing and sharing their emotional or affective state. Thus, it is a form of social communication, which constitutes the most common developmental impairment across autism spectrum disorders (ASD). METHODS: Here we describe the development of a zebrafish model that identifies the neurocognitive mechanisms by which shank3 mutation drives deficits in social contagion. We used a CRISPR-Cas9 technique to generate mutations to the shank3a gene, a zebrafish paralogue found to present greater orthology and functional conservation relative to the human gene. Mutants were first compared to wild types during a two-phase protocol that involves the observation of two conflicting states, distress and neutral, and the later recall and discrimination of others when no longer presenting such differences. Then, the whole-brain expression of different neuroplasticity markers was compared between genotypes and their contribution to cluster-specific phenotypic variation was assessed. RESULTS: The shank3 mutation markedly reduced social contagion via deficits in attention contributing to difficulties in recognising affective states. Also, the mutation changed the expression of neuronal plasticity genes. However, only downregulated neuroligins clustered with shank3a expression under a combined synaptogenesis component that contributed specifically to variation in attention. LIMITATIONS: While zebrafish are extremely useful in identifying the role of shank3 mutations to composite social behaviour, they are unlikely to represent the full complexity of socio-cognitive and communication deficits presented by human ASD pathology. Moreover, zebrafish cannot represent the scaling up of these deficits to higher-order empathic and prosocial phenotypes seen in humans. CONCLUSIONS: We demonstrate a causal link between the zebrafish orthologue of an ASD-associated gene and the attentional control of affect recognition and consequent social contagion. This models autistic affect-communication pathology in zebrafish and reveals a genetic attention-deficit mechanism, addressing the ongoing debate for such mechanisms accounting for emotion recognition difficulties in autistic individuals.

Social zebrafish: Danio rerio as an emerging model in social neuroendocrinology.

The fitness benefits of social life depend on the ability of animals to affiliate with others and form groups, on dominance hierarchies within groups that determine resource distribution, and on cognitive capacities for recognition, learning and information transfer. The evolution of these phenotypes is coupled with that of neuroendocrine mechanisms, but the causal link between the two remains underexplored. Growing evidence from our research group and others demonstrates that the tools available in zebrafish, Danio rerio, can markedly facilitate progress in this field. Here, we review this evidence and provide a synthesis of the state-of-the-art in this model system. We discuss the involvement of generalized motivation and cognitive components, neuroplasticity and functional connectivity across social decision-making brain areas, and how these are modulated chiefly by the oxytocin-vasopressin neuroendocrine system, but also by reward-pathway monoamine signaling and the effects of sex-hormones and stress physiology.

Evolutionarily conserved role of oxytocin in social fear contagion in zebrafish.

Emotional contagion is the most ancestral form of empathy. We tested to what extent the proximate mechanisms of emotional contagion are evolutionarily conserved by assessing the role of oxytocin, known to regulate empathic behaviors in mammals, in social fear contagion in zebrafish. Using oxytocin and oxytocin receptor mutants, we show that oxytocin is both necessary and sufficient for observer zebrafish to imitate the distressed behavior of conspecific demonstrators. The brain regions associated with emotional contagion in zebrafish are homologous to those involved in the same process in rodents (e.g., striatum, lateral septum), receiving direct projections from oxytocinergic neurons located in the pre-optic area. Together, our results support an evolutionary conserved role for oxytocin as a key regulator of basic empathic behaviors across vertebrates.

Social Enhancement of Adult Neurogenesis in Zebrafish is Not Regulated by Cortisol.

In Mammals adult neurogenesis is influenced by environmental conditions, and the glucocorticoid hormones (GC) play a major role in this regulation. In contrast in fish, the study of the effects of cortisol on the regulation of environmental driven adult neurogenesis has produced conflicting results. While in some species elevated cortisol levels impair cell proliferation, in others, it promotes cell proliferation and differentiation. This lack of consistency may be explained by methodological differences across studies, namely in the stimuli and/or cortisol treatments used. Here, we tested the effects of the social environment on adult neurogenesis, considering a positive and a negative social context, and different durations of cortisol exposure. We hypothesise that there is an interaction between the valence of the social environment and cortisol, such that elevated acute cortisol experienced during social interactions only have a detrimental effect on neurogenesis in negative social contexts. Therefore, fish were exposed to a positive (conspecific shoal) or negative (predator) social experience, and the interaction between the valence of the social context and cortisol exposure (acute and chronic) was tested. Our results indicate that adult neurogenesis is modulated by the social environment, with the number of newly generated cells being dependent on the valence of the social information (positive > negative). These effects were independent of cortisol, either for acute or chronic exposure, highlighting the social environment as a key factor in the modulation of cell proliferation in the adult zebrafish brain, and rejecting a role for cortisol in this modulation.

Phenotypic architecture of sociality and its associated genetic polymorphisms in zebrafish.

Sociality relies on motivational and cognitive components that may have evolved independently, or may have been linked by phenotypic correlations driven by a shared selective pressure for increased social competence. Furthermore, these components may be domain-specific or of general-domain across social and non-social contexts. Here, we used zebrafish to test if the motivational and cognitive components of social behavior are phenotypically linked and if they are domain specific or of general domain. The behavioral phenotyping of zebrafish in social and equivalent non-social tests shows that the motivational (preference) and cognitive (memory) components of sociality: (1) are independent from each other, hence not supporting the occurrence of a sociality syndrome; and (2) are phenotypically linked to non-social traits, forming two general behavioral modules, suggesting that sociality traits have been co-opted from general-domain motivational and cognitive traits. Moreover, the study of the association between single nucleotide polymorphisms (SNPs) and each behavioral module further supports this view, since several SNPs from a list of candidate "social" genes, are statistically associated with the motivational, but not with the cognitive, behavioral module. Together, these results support the occurrence of general-domain motivational and cognitive behavioral modules in zebrafish, which have been co-opted for the social domain.

Social stimuli increase activity of adult-born cells in the telencephalon of zebrafish (Danio rerio).

Fish have particularly high levels of adult neurogenesis, and this high neurogenic capacity may contribute to behavioural plasticity. While it is known that adult-born cells can differentiate into neurons and incorporate into neural circuits, it is unclear whether they are responsive to external stimuli and are thereby capable of contributing to behavioural change. We tested whether cells born in the telencephalon of adult zebrafish are activated by social stimuli. We marked cell birth with BrdU and, 40 days later, exposed fish to brief (15 min) visual social stimuli and assayed cellular activity through immunolocalization of phospho-S6-ribosomal protein (pS6). BrdU+/pS6+ co-labelled cells were found in six brain regions, and, in four regions [dorsal (D), dorsomedial (Dm) and dorsolateral (Dl) zones of the dorsal telencephalon and pre-optic area (POA)], the number of co-labelled cells and fraction of BrdU+ cells that labelled positive for pS6 increased during social stimulation. These results are consistent with the hypothesis that adult-born neurons play a role in regulating social behaviour.

Early social deprivation shapes neuronal programming of the social decision-making network in a cooperatively breeding fish.

The early social environment an animal experiences may have pervasive effects on its behaviour. The social decision-making network (SDMN), consisting of interconnected brain nuclei from the forebrain and midbrain, is involved in the regulation of behaviours during social interactions. In species with advanced sociality such as cooperative breeders, offspring are exposed to a large number and a great diversity of social interactions every day of their early life. This diverse social environment may have life-long consequences on the development of several neurophysiological systems within the SDMN, although these effects are largely unknown. We studied these life-long effects in a cooperatively breeding fish, Neolamprologus pulcher, focusing on the expression of genes involved in the monoaminergic and stress response systems in the SDMN. N. pulcher fry were raised until an age of 2 months either with their parents, subordinate helpers and same-clutch siblings (+F), or with same-clutch siblings only (-F). Analysis of the expression of glucocorticoid receptor, mineralocorticoid receptor, corticotropin releasing factor, dopamine receptors 1 and 2, serotonin transporter and DNA methyltransferase 1 genes showed that early social experiences altered the neurogenomic profile of the preoptic area. Moreover, the dopamine receptor 1 gene was up-regulated in the preoptic area of -F fish compared to +F fish. -F fish also showed up-regulation of GR1 expression in the dorsal medial telencephalon (functional equivalent to the basolateral amygdala), and in the dorsolateral telencephalon (functional equivalent to the hippocampus). Our results suggest that early social environment has life-long effects on the development of several neurophysiological systems within the SDMN.

Brain morphology predicts social intelligence in wild cleaner fish.

It is generally agreed that variation in social and/or environmental complexity yields variation in selective pressures on brain anatomy, where more complex brains should yield increased intelligence. While these insights are based on many evolutionary studies, it remains unclear how ecology impacts brain plasticity and subsequently cognitive performance within a species. Here, we show that in wild cleaner fish (Labroides dimidiatus), forebrain size of high-performing individuals tested in an ephemeral reward task covaried positively with cleaner density, while cerebellum size covaried negatively with cleaner density. This unexpected relationship may be explained if we consider that performance in this task reflects the decision rules that individuals use in nature rather than learning abilities: cleaners with relatively larger forebrains used decision-rules that appeared to be locally optimal. Thus, social competence seems to be a suitable proxy of intelligence to understand individual differences under natural conditions.

Quantifying Aggressive Behavior in Zebrafish.

Aggression is a complex behavior that influences social relationships and can be seen as adaptive or maladaptive depending on the context and intensity of expression. A model organism suitable for genetic dissection of the underlying neural mechanisms of aggressive behavior is still needed. Zebrafish has already proven to be a powerful vertebrate model organism for the study of normal and pathological brain function. Despite the fact that zebrafish is a gregarious species that forms shoals, when allowed to interact in pairs, both males and females express aggressive behavior and establish dominance hierarchies. Here, we describe two protocols that can be used to quantify aggressive behavior in zebrafish, using two different paradigms: (1) staged fights between real opponents and (2) mirror-elicited fights. We also discuss the methodology for the behavior analysis, the expected results for both paradigms, and the advantages and disadvantages of each paradigm in face of the specific goals of the study.

Agonistic interactions elicit rapid changes in brain nonapeptide levels in zebrafish.

The teleost fish nonapeptides, arginine vasotocin (AVT) and isotocin (IT), have been implicated in the regulation of social behavior. These peptides are expected to be involved in acute and transient changes in social context, in order to be efficient in modulating the expression of social behavior according to changes in the social environment. Here we tested the hypothesis that short-term social interactions are related to changes in the level of both nonapeptides across different brain regions. For this purpose we exposed male zebrafish to two types of social interactions: (1) real opponent interactions, from which a Winner and a Loser emerged; and (2) mirror-elicited interactions, that produced individuals that did not experience a change in social status despite expressing similar levels of aggressive behavior to those of participants in real-opponent fights. Non-interacting individuals were used as a reference group. Each social phenotype (i.e. Winners, Losers, Mirror-fighters) presented a specific brain profile of nonapeptides when compared to the reference group. Moreover, the comparison between the different social phenotypes allowed to address the specific aspects of the interaction (e.g. assessment of opponent aggressive behavior vs. self-assessment of expressed aggressive behavior) that are linked with neuropeptide responses. Overall, agonistic interactions seem to be more associated with the changes in brain AVT than IT, which highlights the preferential role of AVT in the regulation of aggressive behavior already described for other species.

Social Plasticity Relies on Different Neuroplasticity Mechanisms across the Brain Social Decision-Making Network in Zebrafish.

Social living animals need to adjust the expression of their behavior to their status within the group and to changes in social context and this ability (social plasticity) has an impact on their Darwinian fitness. At the proximate level social plasticity must rely on neuroplasticity in the brain social decision-making network (SDMN) that underlies the expression of social behavior, such that the same neural circuit may underlie the expression of different behaviors depending on social context. Here we tested this hypothesis in zebrafish by characterizing the gene expression response in the SDMN to changes in social status of a set of genes involved in different types of neural plasticity: bdnf, involved in changes in synaptic strength; npas4, involved in contextual learning and dependent establishment of GABAergic synapses; neuroligins (nlgn1 and nlgn2) as synaptogenesis markers; and genes involved in adult neurogenesis (wnt3 and neurod). Four social phenotypes were experimentally induced: Winners and Losers of a real-opponent interaction; Mirror-fighters, that fight their own image in a mirror and thus do not experience a change in social status despite the expression of aggressive behavior; and non-interacting fish, which were used as a reference group. Our results show that each social phenotype (i.e., Winners, Losers, and Mirror-fighters) present specific patterns of gene expression across the SDMN, and that different neuroplasticity genes are differentially expressed in different nodes of the network (e.g., BDNF in the dorsolateral telencephalon, which is a putative teleost homolog of the mammalian hippocampus). Winners expressed unique patterns of gene co-expression across the SDMN, whereas in Losers and Mirror-fighters the co-expression patterns were similar in the dorsal regions of the telencephalon and in the supracommissural nucleus of the ventral telencephalic area, but differents in the remaining regions of the ventral telencephalon. These results indicate that social plasticity relies on multiple neuroplasticity mechanisms across the SDMN, and that there is not a single neuromolecular module underlying this type of behavioral flexibility.

Assessment of fight outcome is needed to activate socially driven transcriptional changes in the zebrafish brain.

Group living animals must be able to express different behavior profiles depending on their social status. Therefore, the same genotype may translate into different behavioral phenotypes through socially driven differential gene expression. However, how social information is translated into a neurogenomic response and what are the specific cues in a social interaction that signal a change in social status are questions that have remained unanswered. Here, we show for the first time, to our knowledge, that the switch between status-specific neurogenomic states relies on the assessment of fight outcome rather than just on self- or opponent-only assessment of fighting ability. For this purpose, we manipulated the perception of fight outcome in male zebrafish and measured its impact on the brain transcriptome using a zebrafish whole genome gene chip. Males fought either a real opponent, and a winner and a loser were identified, or their own image on a mirror, in which case, despite expressing aggressive behavior, males did not experience either a victory or a defeat. Massive changes in the brain transcriptome were observed in real opponent fighters, with losers displaying both a higher number of differentially expressed genes and of coexpressed gene modules than winners. In contrast, mirror fighters expressed a neurogenomic state similar to that of noninteracting fish. The genes that responded to fight outcome included immediate early genes and genes involved in neuroplasticity and epigenetic modifications. These results indicate that, even in cognitively simple organisms such as zebrafish, neurogenomic responses underlying changes in social status rely on mutual assessment of fighting ability.

Androgen response to social competition in a shoaling fish.

Androgens respond to social challenges and this response has been interpreted as a way for males to adjust androgen-dependent behavior to social context. However, the androgen responsiveness to social challenges varies across species and a conceptual framework has been developed to explain this variation according to differences in the mating system and parental care type, which determines the regimen of challenges males are exposed to, and concomitantly the scope (defined as the difference between the physiological maximum and the baseline levels) of response to a social challenge. However, this framework has been focused on territorial species and no clear predictions have been made to gregarious species (e.g. shoaling fish), which although tolerating same-sex individuals may also exhibit intra-sexual competition. In this paper we extend the scope of this conceptual framework to shoaling fish by studying the endocrine response of zebrafish (Danio rerio) to social challenges. Male zebrafish exposed to real opponent agonistic interactions exhibited an increase in androgen levels (11-ketotestosterone both in Winners and Losers and testosterone in Losers). This response was absent in Mirror-fighters, that expressed similar levels of aggressive behavior to those of winners, suggesting that this response is not a mere reflex of heightened aggressive motivation. Cortisol levels were also measured and indicated an activation of the hypothalamic-pituitary-interrenal axis in Winners of real opponent fighters, but not Losers or in Mirror-fighters. These results confirm that gregarious species also exhibit an endocrine response to an acute social challenge.

Social interactions elicit rapid shifts in functional connectivity in the social decision-making network of zebrafish.

According to the social decision-making (SDM) network hypothesis, SDM is encoded in a network of forebrain and midbrain structures in a distributed and dynamic fashion, such that the expression of a given social behaviour is better reflected by the overall profile of activation across the different loci rather than by the activity of a single node. This proposal has the implicit assumption that SDM relies on integration across brain regions, rather than on regional specialization. Here we tested the occurrence of functional localization and of functional connectivity in the SDM network. For this purpose we used zebrafish to map different social behaviour states into patterns of neuronal activity, as indicated by the expression of the immediate early genes c-fos and egr-1, across the SDM network. The results did not support functional localization, as some loci had similar patterns of activity associated with different social behaviour states, and showed socially driven changes in functional connectivity. Thus, this study provides functional support to the SDM network hypothesis and suggests that the neural context in which a given node of the network is operating (i.e. the state of its interconnected areas) is central to its functional relevance.

Neurogenomic mechanisms of social plasticity.

Group-living animals must adjust the expression of their social behaviour to changes in their social environment and to transitions between life-history stages, and this social plasticity can be seen as an adaptive trait that can be under positive selection when changes in the environment outpace the rate of genetic evolutionary change. Here, we propose a conceptual framework for understanding the neuromolecular mechanisms of social plasticity. According to this framework, social plasticity is achieved by rewiring or by biochemically switching nodes of a neural network underlying social behaviour in response to perceived social information. Therefore, at the molecular level, it depends on the social regulation of gene expression, so that different genomic and epigenetic states of this brain network correspond to different behavioural states, and the switches between states are orchestrated by signalling pathways that interface the social environment and the genotype. Different types of social plasticity can be recognized based on the observed patterns of inter- versus intra-individual occurrence, time scale and reversibility. It is proposed that these different types of social plasticity rely on different proximate mechanisms at the physiological, neural and genomic level.

Oestradiol and prostaglandin F2α regulate sexual displays in females of a sex-role reversed fish.

The mechanisms regulating sexual behaviours in female vertebrates are still poorly understood, mainly because in most species sexual displays in females are more subtle and less frequent than displays in males. In a sex-role reversed population of a teleost fish, the peacock blenny Salaria pavo, an external fertilizer, females are the courting sex and their sexual displays are conspicuous and unambiguous. We took advantage of this to investigate the role of ovarian-synthesized hormones in the induction of sexual displays in females. In particular, the effects of the sex steroids oestradiol (E2) and testosterone (T) and of the prostaglandin F2α (PGF2α) were tested. Females were ovariectomized and their sexual behaviour tested 7 days (sex steroids and PGF2α) and 14 days (sex steroids) after ovariectomy by presenting females to an established nesting male. Ovariectomy reduced the expression of sexual behaviours, although a significant proportion of females still courted the male 14 days after the ovary removal. Administration of PGF2α to ovariectomized females recovered the frequency of approaches to the male's nest and of courtship displays towards the nesting male. However, E2 also had a positive effect on sexual behaviour, particularly on the frequency of approaches to the male's nest. T administration failed to recover sexual behaviours in ovariectomized females. These results suggest that the increase in E2 levels postulated to occur during the breeding season facilitates female mate-searching and assessment behaviours, whereas PGF2α acts as a short-latency endogenous signal informing the brain that oocytes are mature and ready to be spawned. In the light of these results, the classical view for female fishes, that sex steroids maintain sexual behaviour in internal fertilizers and that prostaglandins activate spawning behaviours in external fertilizers, needs to be reviewed.

Social modulation of brain monoamine levels in zebrafish.

In social species animals tend to adjust their social behaviour according to the available social information in the group, in order to optimize and improve their one social status. This changing environment requires for rapid and transient behavioural changes that relies primarily on biochemical switching of existing neural networks. Monoamines and neuropeptides are the two major candidates to mediate these changes in brain states underlying socially behavioural flexibility. In the current study we used zebrafish (Danio rerio) males to study the effects of acute social interactions on rapid regional changes in brain levels of monoamines (serotonin and dopamine). A behavioural paradigm under which male zebrafish consistently express fighting behaviour was used to investigate the effects of different social experiences: winning the interaction, losing the interaction, or fighting an unsolved interaction (mirror image). We found that serotonergic activity is significantly higher in the telencephalon of winners and in the optic tectum of losers, and no significant changes were observed in mirror fighters suggesting that serotonergic activity is differentially regulated in different brain regions by social interactions. Dopaminergic activity it was also significantly higher in the telencephalon of winners which may be representative of social reward. Together our data suggests that acute social interactions elicit rapid and differential changes in serotonergic and dopaminergic activity across different brain regions.

A three-dimensional stereotaxic MRI brain atlas of the cichlid fish Oreochromis mossambicus.

The African cichlid Oreochromis mossambicus (Mozambique tilapia) has been used as a model system in a wide range of behavioural and neurobiological studies. The increasing number of genetic tools available for this species, together with the emerging interest in its use for neurobiological studies, increased the need for an accurate hodological mapping of the tilapia brain to supplement the available histological data. The goal of our study was to elaborate a three-dimensional, high-resolution digital atlas using magnetic resonance imaging, supported by Nissl staining. Resulting images were viewed and analysed in all orientations (transverse, sagittal, and horizontal) and manually labelled to reveal structures in the olfactory bulb, telencephalon, diencephalon, optic tectum, and cerebellum. This high resolution tilapia brain atlas is expected to become a very useful tool for neuroscientists using this fish model and will certainly expand their use in future studies regarding the central nervous system.

Adult neurogenesis in the brain of the Mozambique tilapia, Oreochromis mossambicus.

Although the generation of new neurons in the adult nervous system ('adult neurogenesis') has been studied intensively in recent years, little is known about this phenomenon in non-mammalian vertebrates. Here, we examined the generation, migration, and differentiation of new neurons and glial cells in the Mozambique tilapia (Oreochromis mossambicus), a representative of one of the largest vertebrate taxonomic orders, the perciform fish. The vast majority of new cells in the brain are born in specific proliferation zones of the olfactory bulb; the dorsal and ventral telencephalon; the periventricular nucleus of the posterior tuberculum, optic tectum, and nucleus recessi lateralis of the diencephalon; and the valvula cerebelli, corpus cerebelli, and lobus caudalis of the cerebellum. As shown in the olfactory bulb and the lateral part of the valvula cerebelli, some of the young cells migrate from their site of origin to specific target areas. Labeling of mitotic cells with the thymidine analog 5-bromo-2'-deoxyuridine, combined with immunostaining against the neuron-specific marker protein Hu or against the astroglial marker glial fibrillary acidic protein demonstrated differentiation of the adult-born cells into both neurons and glia. Taken together, the present investigation supports the hypothesis that adult neurogenesis is an evolutionarily conserved vertebrate trait.