Inhibitory control in teleost fish: a methodological and conceptual review.

Inhibitory control (IC) plays a central role in behaviour control allowing an individual to resist external lures and internal predispositions. While IC has been consistently investigated in humans, other mammals, and birds, research has only recently begun to explore IC in other vertebrates. This review examines current literature on teleost fish, focusing on both methodological and conceptual aspects. I describe the main paradigms adopted to study IC in fish, identifying well-established tasks that fit various research applications and highlighting their advantages and limitations. In the conceptual analysis, I identify two well-developed lines of research with fish examining IC. The first line focuses on a comparative approach aimed to describe IC at the level of species and to understand the evolution of interspecific differences in relation to ecological specialisation, brain size, and factors affecting cognitive performance. Findings suggest several similarities between fish and previously studied vertebrates. The second line of research focuses on intraspecific variability of IC. Available results indicate substantial variation in fish IC related to sex, personality, genetic, age, and phenotypic plasticity, aligning with what is observed with other vertebrates. Overall, this review suggests that although data on teleosts are still scarce compared to mammals, the contribution of this group to IC research is already substantial and can further increase in various disciplines including comparative psychology, cognitive ecology, and neurosciences, and even in applied fields such as psychiatry research.

Adaptive phenotypic plasticity induces individual variability along a cognitive trade-off.

Animal species, including humans, display patterns of individual variability in cognition that are difficult to explain. For instance, some individuals perform well in certain cognitive tasks but show difficulties in others. We experimentally analysed the contribution of cognitive plasticity to such variability. Theory suggests that diametrically opposed cognitive phenotypes increase individuals' fitness in environments with different conditions such as resource predictability. Therefore, if selection has generated plasticity that matches individuals' cognitive phenotypes to the environment, this might produce remarkable cognitive variability. We found that guppies, Poecilia reticulata, exposed to an environment with high resource predictability (i.e. food available at the same time and in the same location) developed enhanced learning abilities. Conversely, guppies exposed to an environment with low resource predictability (i.e. food available at a random time and location) developed enhanced cognitive flexibility and inhibitory control. These cognitive differences align along a trade-off between functions that favour the acquisition of regularities such as learning and functions that adjust behaviour to changing conditions (cognitive flexibility and inhibitory control). Therefore, adaptive cognitive plasticity in response to resource predictability (and potentially similar factors) is a key determinant of cognitive individual differences.

Interspecific differences in developmental mode determine early cognitive abilities in teleost fish.

Most studies on developmental variation in cognition have suggested that individuals are born with reduced or absent cognitive abilities, and thereafter, cognitive performance increases with age during early development. However, these studies have been mainly performed in altricial species, such as humans, in which offspring are extremely immature at birth. In this work, we tested the hypothesis that species with other developmental modes might show different patterns of cognitive development. To this end, we analysed inhibitory control performance in two teleost species with different developmental modes, the zebrafish Danio rerio and the guppy Poecilia reticulata, exploiting a simple paradigm based on spontaneous behaviour and therefore applicable to subjects of different ages. Zebrafish hatch as larvae 3 days after fertilisation, and have an immature nervous system, a situation that mirrors extreme altriciality. We found that at the early stages of development, zebrafish displayed no evidence of inhibitory control, which only begun to emerge after one month of life. Conversely, guppies, which are born after approximately one month of gestation as fully developed and independent individuals, solved the inhibitory control task since their first days of life, although performance increased with sexual maturation. Our study suggests that the typical progression described during early ontogeny in humans and other species might not be the only developmental trend for animals' cognition and that a species' developmental mode might determine variation in cognition across subjects of different age.

Embryonic exposure to artificial light at night impairs learning abilities and their covariance with behavioural traits in teleost fish.

The natural light cycle has profound effects on animals' cognitive systems. Its alteration owing to human activities, such as artificial light at night (ALAN), affects the biodiversity of mammalian and avian species by impairing their cognitive functions. The impact of ALAN on cognition, however, has not been investigated in aquatic species, in spite of the common occurrence of this pollution along water bodies. We exposed eggs of a teleost fish (the zebrafish Danio rerio) to ALAN and, upon hatching, we measured larvae' cognitive abilities with a habituation learning paradigm. Both control and ALAN-exposed larvae showed habituation learning, but the latter learned significantly slower, suggesting that under ALAN conditions, fish require many more events to acquire ecologically relevant information. We also found that individuals' learning performance significantly covaried with two behavioural traits in the control zebrafish, but ALAN disrupted one of these relationships. Additionally, ALAN resulted in an average increase in larval activity. Our results showed that both fish's cognitive abilities and related individual differences are negatively impacted by light pollution, even after a short exposure in the embryonic stage.

Modeling Sarcoglycanopathy in Danio rerio.

Sarcoglycanopathies, also known as limb girdle muscular dystrophy 3-6, are rare muscular dystrophies characterized, although heterogeneous, by high disability, with patients often wheelchair-bound by late adolescence and frequently developing respiratory and cardiac problems. These diseases are currently incurable, emphasizing the importance of effective treatment strategies and the necessity of animal models for drug screening and therapeutic verification. Using the CRISPR/Cas9 genome editing technique, we generated and characterized δ-sarcoglycan and ß-sarcoglycan knockout zebrafish lines, which presented a progressive disease phenotype that worsened from a mild larval stage to distinct myopathic features in adulthood. By subjecting the knockout larvae to a viscous swimming medium, we were able to anticipate disease onset. The δ-SG knockout line was further exploited to demonstrate that a δ-SG missense mutant is a substrate for endoplasmic reticulum-associated degradation (ERAD), indicating premature degradation due to protein folding defects. In conclusion, our study underscores the utility of zebrafish in modeling sarcoglycanopathies through either gene knockout or future knock-in techniques. These novel zebrafish lines will not only enhance our understanding of the disease's pathogenic mechanisms, but will also serve as powerful tools for phenotype-based drug screening, ultimately contributing to the development of a cure for sarcoglycanopathies.

Individual differences and knockout in zebrafish reveal similar cognitive effects of BDNF between teleosts and mammals.

The remarkable similarities in cognitive performance between teleosts and mammals suggest that the underlying cognitive mechanisms might also be similar in these two groups. We tested this hypothesis by assessing the effects of the brain-derived neurotrophic factor (BDNF), which is critical for mammalian cognitive functioning, on fish's cognitive abilities. We found that individual differences in zebrafish's learning abilities were positively correlated with bdnf expression. Moreover, a CRISPR/Cas9 mutant zebrafish line that lacks the BDNF gene (bdnf-/-) showed remarkable learning deficits. Half of the mutants failed a colour discrimination task, whereas the remaining mutants learned the task slowly, taking three times longer than control bdnf+/+ zebrafish. The mutants also took twice as long to acquire a T-maze task compared to control zebrafish and showed difficulties exerting inhibitory control. An analysis of habituation learning revealed that cognitive impairment in mutants emerges early during development, but could be rescued with a synthetic BDNF agonist. Overall, our study indicates that BDNF has a similar activational effect on cognitive performance in zebrafish and in mammals, supporting the idea that its function is conserved in vertebrates.

Medaka as a model for seasonal plasticity: Photoperiod-mediated changes in behaviour, cognition, and hormones.

Teleosts display the highest level of brain plasticity of all vertebrates. Yet we still know little about how seasonality affects fish behaviour and the underlying cognitive mechanisms since the common neurobehavioral fish models are native to tropical environments where seasonal variation is absent or reduced. The medaka, Oryzias latipes, which inhabits temperate zone habitats, represents a promising model in this context given its large phenotypic changes associated with seasonality and the possibility to induce seasonal plasticity by only manipulating photoperiod. Here, we report the first extended investigation of seasonal plasticity in medaka behaviour and cognition, as well as the potential underlying molecular mechanisms. We compared medaka exposed to summer photoperiod (16 h light:8 h dark) with medaka exposed to winter photoperiod (8 h light:16 h dark), and detected substantial differences. Medaka were more active and less social in summer photoperiod conditions, two effects that emerged in the second half of an open-field and a sociability test, respectively, and might be at least in part related to habituation to the testing apparatus. Moreover, the cognitive phenotype was significantly affected: in the early response to a social stimulus, brain functional lateralisation shifted between the two hemispheres under the two photoperiod conditions, and inhibitory and discrimination learning performance were reduced in summer conditions. Finally, the expression of genes encoding key pituitary hormones, tshß and gh, and of the tshß regulatory transcription factor tef in the brain was increased in summer photoperiod conditions. This work reveals remarkable behavioural and cognitive phenotypic plasticity in response to photoperiod in medaka, and suggests a potential regulatory role for the same hormones involved in seasonal plasticity of other vertebrates.

Environmental enrichment decreases anxiety-like behavior in zebrafish larvae.

The development of anxiety disorders is often linked to individuals' negative experience. In many animals, development of anxiety-like behavior is modeled by manipulating individuals' exposure to environmental enrichment. We investigated whether environmental enrichment during early ontogenesis affects anxiety-like behavior in larval zebrafish. Larvae were exposed from hatching to either an environment enriched with 3D-objects of different color and shape or to a barren environment. Behavioral testing was conducted at different intervals during development (7, 14, and 21 days post-fertilization, dpf). In a novel object exploration test, 7 dpf larvae of the two treatments displayed similar avoidance of the visual stimulus. However, at 14 and 21 dpf, larvae of the enriched environment showed less avoidance, indicating lower anxiety response. Likewise, larvae of the two treatments demonstrated comparable avoidance of a novel odor stimulus at 7 dpf, with a progressive reduction of anxiety behavior in the enriched treatment with development. In a control experiment, larvae treated before 7 dpf but tested at 14 dpf showed the effect of enrichment on anxiety, suggesting an early determination of the anxiety phenotype. This study confirms a general alteration of zebrafish anxiety-like behavior due to a short enrichment period in first days of life.

Behavioural effects of early-life exposure to parabens in zebrafish larvae.

Parabens are classified as endocrine disrupting chemicals due to their ability to activate several nuclear receptors causing changes in hormones-dependent signalling pathways. Central nervous system of developing organisms is particularly vulnerable to changes in hormonal pathways, which could lead to altered brain function, abnormal behaviour and even diseases later in life. The aim of the present study was to investigate the effects of exposure to butylparaben (BuP), ethylparaben (EtP) and methylparaben (MeP) during early development on nervous system using zebrafish larvae's behavioural models. Zebrafish were exposed until 4 days post fertilization (dpf) to three concentrations of each paraben chosen considering the environmentally realistic concentrations of human exposure and the benchmark-dose lower bound calculated for zebrafish larvae (BuP: 5, 50 and 500 µg/L; EtP: 50, 500 and 5000 µg/L; MeP: 100, 1000 and 10,000 µg/L). Activity in novel and in familiar environment, thigmotaxis, visual startle response and photic synchronization of the behavioural circadian rhythms were analysed at 4, 5 and 6 dpf. Zebrafish larvae exposed to BuP 500 µg/L and EtP 5000 µg/L revealed increased anxiety-like behaviour in novel environment. Larvae treated with 500 µg/L of BuP showed reduced activity in familiar and marginally in unfamiliar environment, and larvae exposed to 5000 µg/L of EtP exhibited hyperactivity in familiar environment. Parabens exposure did not influence the visual startle response and the photic synchronization of circadian rhythms in zebrafish larvae. This research highlighted as the exposure to parabens has the potential to interfere with behavioural development of zebrafish.

Guppies show sex and individual differences in the ability to inhibit behaviour.

In humans, individual and sex differences have been long reported for several cognitive tasks and are at least in part due to variability in the function that inhibits behaviour (i.e. inhibitory control). Similar evidence of individual and sex differences in inhibitory abilities is also present in other vertebrates, but is scarce outside primates. Experiments on reversal learning, which requires inhibiting behaviours, suggest that this variability may exist in a teleost fish, the guppy, Poecilia reticulata. We tested this hypothesis by observing guppies in an inhibitory task. Guppies were exposed to unreachable prey inside a transparent tube for six trials. Guppies showed a marked reduction in the number of attempts to catch the prey within the first trial and also over repeated trials. We found a striking sex difference in the capacity to inhibit foraging behaviour. Males attempted to attack the prey twice as often as females and showed negligible improvement over trials. Irrespective of sex, individuals remarkably differed in their performance, with some guppies being systematically more skilled than others across the repeated trials. These results confirm that individual and sex differences in the ability to inhibit behaviour are not restricted to humans and other primates, suggesting that they might be widespread among vertebrates. Variability in inhibitory ability provides an explanation for emerging records of variability in other cognitive tasks in fish.

Lateralization correlates with individual differences in inhibitory control in zebrafish.

Individual fitness often depends on the ability to inhibit behaviours not adapted to a given situation. However, inhibitory control can vary greatly between individuals of the same species. We investigated a mechanism that might maintain this variability in zebrafish (Danio rerio). We demonstrate that inhibitory control correlates with cerebral lateralization, the tendency to process information with one brain hemisphere or the other. Individuals that preferentially observed a social stimulus with the right eye and thus processed social information with the left brain hemisphere, inhibited foraging behaviour more efficiently. Therefore, selective pressures that maintain lateralization variability in populations might provide indirect selection for variability in inhibitory control. Our study suggests that individual cognitive differences may result from complex multi-trait selection mechanisms.

Inhibitory control in zebrafish, Danio rerio.

We assessed whether zebrafish, Danio rerio, display inhibitory control using a simple and rapid behavioural test. Zebrafish were exposed to a prey stimulus placed inside a transparent tube, which initially elicited attack behaviour. However, zebrafish showed a rapid reduction in the number of attacks towards the prey, which indicated the ability to inhibit their foraging behaviour. Zebrafish also exhibited mnemonic retention of foraging inhibition, as indicated by a reduced number of attacks in a subsequent exposure to the unreachable prey. The ability to inhibit the foraging behaviour varied across three genetically separated wild-type strains and across different individuals within strains, suggesting that zebrafish show heritable within-species differences in inhibitory control. Our behavioural test might be suitable for screening large zebrafish populations in mutational studies and assessing the effects of pharmacologically active substances on inhibitory control.

Guppies, Poecilia reticulata, perceive a reversed Delboeuf illusion.

Animals are often required to estimate object sizes during several fitness-related activities, such as choosing mates, foraging, and competing for resources. Some species are susceptible to size illusions, i.e. the misperception of the size of an object based on the surrounding context, but other species are not. This interspecific variation might be adaptive, reflecting species-specific selective pressures; according to this hypothesis, it is important to test species in which size discrimination has a notable ecological relevance. We tested susceptibility to a size illusion in the guppy, Poecilia reticulata, a fish species required to accurately estimate sizes during mate choice, foraging, and antipredator behaviours. We focussed on the Delboeuf illusion, in which an object is typically perceived to be larger when surrounded by a smaller object. In experiment 1, we trained guppies to select the larger of two circles to obtain a food reward and then tested them using stimuli arranged in a Delboeuf-like pattern. In experiment 2, we tested guppies in a spontaneous food choice task to determine whether the subjective size perception of food items is affected by the surrounding context. Jointly, our experiments indicated that guppies perceived the Delboeuf illusion, but in a reverse direction relative to humans: guppies estimated as larger the stimulus that human perceived as smaller. Our results indicated susceptibility to size illusions also in a species required to perform accurate size discrimination and support previous evidence of variability in illusion susceptibility across vertebrates.

Familiarity and sex modulate size-dependent following behaviour in the Mediterranean killifish.

Living in a group offers the chance to follow the choices and the behaviours of other individuals. Following a group mate might confer fitness advantages if the group mate knows about resources such as food or shelters. Shoaling fish often follow larger group mates which, in most species, are generally older and therefore more experienced. Yet, the effect of individuals' characteristics other than size on following behaviour remains to be understood. For example, familiar fish and female shoals have been reported as more cohesive, which might be due to a differential tendency to follow in relation to familiarity and sex. Here, we investigated whether size, familiarity, sex, and the interaction between these factors affect fish following behaviour. We observed pairs of differently sized Mediterranean killifish, Aphanius fasciatus, exploring a new environment, and we recorded whether the rear fish followed the front fish when the latter changed swimming direction. In female and male pairs, and in unfamiliar pairs, smaller fish were more likely to follow the directional change of the larger fish than vice versa. In mixed-sex pairs and in familiar pairs, however, size did not affect following behaviour and larger fish followed as much as smaller fish did. Our results revealed that killifish's following decisions are determined by the size of the individuals, their level of familiarity, and their sex. These characteristics may have a notable impact on the behaviour of fish groups in nature.

Development and testing of a rapid method for measuring shoal size discrimination.

The shoal-choice test is a popular method to investigate quantity discrimination in social fish based on their spontaneous preference for the larger of two shoals. The shoal-choice test usually requires a long observation time (20-30 min), mainly because fish switch between the two shoals with low frequency, thus reducing the possibilities of comparison. This duration limits the use of the shoal-choice test for large-scale screenings. Here, we developed a new version of the shoal-choice test in which the subject was confined in a large transparent cylinder in the middle of the tank throughout the experiment to bound the minimum distance from the stimulus shoals and favour switching. We tested the new method by observing guppies (Poecilia reticulata) in a 4 versus 6 fish discrimination (experiment 1). The new method allowed for a faster assessment of the preference for the larger shoal (<5 min), resulting in potential application for large population screenings. Guppies switched five times more frequently between the two shoals and remained close to the first chosen shoal ten times less compared to experiments with the old method. In experiment 2, we found that with the new method guppies were able to discriminate up to 5 versus 6 fish, a discrimination that was not achieved with the classical method. This last result indicates that minor methodological modifications can lead to very different findings in the same species and suggests that caution should be exercised when interpreting inter-specific differences in quantitative abilities.

Sex differences in discrimination reversal learning in the guppy.

In several mammalian and avian species, females show a higher performance than males in tasks requiring cognitive flexibility such as the discrimination reversal learning. A recent study showed that female guppies are twice as efficient as males in a reversal learning task involving yellow-red discrimination, suggesting a higher cognitive flexibility in female guppies. However, the possibility exists that the superior performance exhibited by females does not reflect a general sex difference in cognitive abilities, but instead, is confined to colour discrimination tasks. To address this issue, we compared male and female guppies in two different discrimination reversal learning tasks and we performed a meta-analysis of these experiments and the previous one involving colour discrimination. In the first experiment of this study, guppies were tested in a task requiring them to learn to select the correct arm of a T-maze in order to rejoin a group of conspecifics. In experiment 2, guppies were observed in a numerical task requiring them to discriminate between 5 and 10 dots in order to obtain a food reward. Although females outperformed males in one condition of the T-maze, we did not find any clear evidence of females' greater reversal learning performance in either experiment. However, the meta-analysis of the three experiments supported the hypothesis of females' greater reversal learning ability. Our data do not completely exclude the idea that female guppies have a generally higher cognitive flexibility than males; however, they suggest that the size of this sex difference might depend on the task.

Experimental setting affects the performance of guppies in a numerical discrimination task.

A recent study found that guppies (Poecilia reticulata) can be trained to discriminate 4 versus 5 objects, a numerical discrimination typically achieved only by some mammals and birds. In that study, guppies were required to discriminate between two patches of small objects on the bottom of the tank that they could remove to find a food reward. It is not clear whether this species possesses exceptional numerical accuracy compared with the other ectothermic vertebrates or whether its remarkable performance was due to a specific predisposition to discriminate between differences in the quality of patches while foraging. To disentangle these possibilities, we trained guppies to the same numerical discriminations with a more conventional two-choice discrimination task. Stimuli were sets of dots presented on a computer screen, and the subjects received a food reward upon approaching the set with the larger numerosity. Though the cognitive problem was identical in the two experiments, the change in the experimental setting led to a much poorer performance as most fish failed even the 2 versus 3 discrimination. In four additional experiments, we varied the duration of the decision time, the type of stimuli, the length of training, and whether correction was allowed in order to identify the factors responsible for the difference. None of these parameters succeeded in increasing the performance to the level of the previous study, although the group trained with three-dimensional stimuli learned the easiest numerical task. We suggest that the different results with the two experimental settings might be due to constraints on learning and that guppies might be prepared to accurately estimate patch quality during foraging but not to learn an abstract stimulus-reward association.

Male and female guppies differ in speed but not in accuracy in visual discrimination learning.

In many species, males and females have different reproductive roles and/or differ in their ecological niche. Since in these cases the two sexes often face different cognitive challenges, selection may promote some degree of cognitive differentiation, an issue that has received relatively little attention so far. We investigated the existence of sex differences in visual discrimination learning in the guppy, Poecilia reticulata, a fish species in which females show complex mate choice based on male colour pattern. We tested males and females for their ability to learn a discrimination between two different shapes (experiment 1) and between two identical figures with a different orientation (experiment 2). In experiment 3, guppies were required to select an object of the odd colour in a group of five objects. Colours changed daily, and therefore, the solution for this task was facilitated by concept learning. We found males' and females' accuracy practically overlapped in the three experiments, suggesting that the two sexes have similar discrimination learning abilities. Yet, males showed faster decision time than females without any evident speed-accuracy trade-off. This result indicates the existence of consistent between-sex differences in decision speed perhaps due to impulsivity rather than speed in information processing. Our results align with previous literature, indicating that sex differences in cognitive abilities are the exception rather than the rule, while sex differences in cognitive style, i.e. the way in which an individual faces a cognitive task, are much more common.

Making the dead talk: alarm cue-mediated antipredator behaviour and learning are enhanced when injured conspecifics experience high predation risk.

Due to the costs of antipredator behaviour, prey have the ability to finely modulate their response according to the risk they have experienced, and adjust it over different scales of ecological time. Information on which to base their responses can be obtained from direct experience, but also indirectly from nearby conspecifics. In aquatic environments, alarm cues from injured conspecifics are an important and reliable source of information about current predation risk. We used wood frog tadpoles, Lithobates sylvaticus, to investigate whether prey responses to alarm cues match the level of background predation risk experienced by injured conspecifics. We found that tadpoles exposed to alarm cues from conspecifics raised in a high-risk environment showed a stronger antipredator response and an enhanced learned response to novel predators, when compared with tadpoles exposed to alarm cues from conspecifics raised in a low-risk environment. Alarm cues not only allow prey to cope with an ongoing predation event, but also to adjust their behaviour to match background risk in the environment.

Severe hypoxia impairs lateralization in a marine teleost fish.

In intertidal environments, the recurring hypoxic condition at low tide is one of the main factors affecting fish behaviour, causing broad effects on ecological interactions. We assessed the effects of hypoxia on lateralization (e.g. the tendency to turn left or right), a behaviour related to brain functional asymmetry, which is thought to play a key role in several life history aspects of fish. Using staghorn sculpin (Leptocottus armatus), a benthic fish that typically inhabits the intertidal zone, we found that hypoxia affects behavioural lateralization at the population level. On average, staghorn sculpins showed a distinct preference for right turns under normoxic conditions (>90% oxygen saturation), but an equal probability of turning right or left after exposure to hypoxia for 2 h (20% oxygen saturation). The specific turning preference observed in the staghorn sculpin control population is likely to have an adaptive value, for example in predator-prey interactions by enhancing attack success or survival from predatory attacks. Therefore the alteration of lateralization expressed by staghorn sculpins under hypoxic conditions may have far-reaching implications for species ecology and trophic interactions. Moreover, our work raises the need to study this effect in other species, in which a hypoxia-driven disruption of lateralization could affect a wider range of behaviours, such as social interactions and schooling.