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.

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.

Zebrafish excel in number discrimination under an operant conditioning paradigm.

Numerical discrimination is widespread in vertebrates, but this capacity varies enormously between the different species examined. The guppy (Poecilia reticulata), the only teleost examined following procedures that allow a comparison with the other vertebrates, outperforms amphibians, reptiles and many warm-blooded vertebrates, but it is unclear whether this is a feature shared with the other teleosts or represents a peculiarity of this species. We trained zebrafish (Danio rerio) to discriminate between numbers differing by one unit, varying task difficulty from 2 versus 3 to 5 versus 6 items. Non-numerical variables that covary with number, such as density or area, did not affect performance. Most fish reached learning criterion on all tasks up to 4 versus 5 discrimination with no sex difference in accuracy. Although no individual reached learning criterion in the 5 versus 6 task, performance was significant at the group level, suggesting that this may represent the discrimination threshold for zebrafish. Numerosity discrimination abilities of zebrafish compare to those of guppy, being higher than in some warm-blooded vertebrates, such as dogs, horses and domestic fowl, though lower than in parrots, corvids and primates. Learning rate was similar in a control group trained to discriminate between different-sized shapes, but zebrafish were slightly more accurate when discriminating areas than numbers and males were more accurate than females. At the end of the experiment, fish trained on numbers and controls trained on areas generalized to the reciprocal set of stimuli, indicating they had used a relational strategy to solve these tasks.

Intraspecific variation in inhibitory motor control in guppies, Poecilia reticulata.

Inhibitory control (IC) is the ability to overcome impulsive or prepotent but ineffective responses in favour of more appropriate behaviours. The ability to inhibit internal predispositions or external temptations is vital in coping with a complex and variable world. Traditionally viewed as cognitively demanding and a main component of executive functioning and self-control, IC was historically examined in only a few species of birds and mammals but recently a number of studies has shown that a much wider range of taxa rely on IC. Furthermore, there is growing evidence that inhibitory abilities may vary within species at the population and individual levels owing to genetic and environmental factors. Here we use a detour-reaching task, a standard paradigm to measure motor inhibition in nonhuman animals, to quantify patterns of interindividual variation in IC in wild-descendant female guppies, Poecilia reticulata. We found that female guppies displayed inhibitory performances that were, on average, half as successful as the performances reported previously for other strains of guppies tested in similar experimental conditions. Moreover, we showed consistent individual variation in the ability to inhibit inappropriate behaviours. Our results contribute to the understanding of the evolution of fish cognition and suggest that IC may show considerable variation among populations within a species. Such variation in IC abilities might contribute to individual differences in other cognitive functions such as spatial learning, quantity discrimination or reversal learning.

Food density and preferred quantity: discrimination of small and large numbers in angelfish (Pterophyllum scalare).

Many animal species share the ability to discriminate between sets with different quantity of food items. In fish, this ability has rarely been investigated, although findings have been obtained do indicate a preference, as in other animals, for sets with large over small quantities. The role played by food item size has also been found to be important in the discrimination. However, another potentially important non-numerical variable, food density, has not been investigated. In this study, we examined the influence of density (inter-item distance) in the decision-making process of food discrimination in angelfish (Pterophyllum scalare). In a binary choice task, we kept the number and size of food items constant, but contrasted a set containing food items spaced further apart (sparse set) to another set with food items spaced more closely (dense set). We conducted this analysis with sets in the small (3 vs 3 food items) and in the large number range (5 vs 5 food items) and also varied the specific spatial arrangements of the food items in the sets. Contrary to expectations, angelfish showed a preference for the sparse sets over the dense sets in the five vs five contrasts irrespective of the specific spatial arrangement, but exhibited no preference in case of the three vs three contrasts. Subsequently, we slightly lengthened the inter-item distance in the dense sets, and found preference for the dense over the sparse sets. Last, we further examined the potential effect of spatial configuration of the items in the sets, but found no effect of this latter factor. Overall, these results indicate that higher density of the contrasted food item sets significantly influences choice in angelfish, which prefer denser sets if a clear discriminability of each individual item within the sets is provided.

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.

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.

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.

One-trial odour recognition learning and its underlying brain areas in the zebrafish.

Distinguishing familiar from novel stimuli is critical in many animals' activities, and procedures based on this ability are among the most exploited in translational research in rodents. However, recognition learning and the underlying brain substrates remain unclear outside a few mammalian species. Here, we investigated one-trial recognition learning for olfactory stimuli in a teleost fish using a behavioural and molecular approach. With our behavioural analysis, we found that zebrafish can learn to recognise a novel odour after a single encounter and then, discriminate between this odour and a different one provided that the molecular structure of the cues is relatively differentiated. Subsequently, by expression analysis of immediate early genes in the main brain areas, we found that the telencephalon was activated when zebrafish encountered a familiar odour, whereas the hypothalamus and the optic tectum were activated in response to the novel odour. Overall, this study provided evidence of single-trial spontaneous learning of novel odours in a teleost fish and the presence of multiple neural substrates involved in the process. These findings are promising for the development of zebrafish models to investigate cognitive functions.

Environmental conditions shape learning in larval zebrafish.

Growing evidence reveals notable phenotypic plasticity in cognition among teleost fishes. One compelling example is the positive impact of enriched environments on learning performance. Most studies on this effect have focused on juvenile or later life stages, potentially overlooking the importance of early life plasticity. To address this gap, we investigated whether cognitive plasticity in response to environmental factors emerges during the larval stage in zebrafish. Our findings indicate that larvae exposed to an enriched environment after hatching exhibited enhanced habituation learning performance compared to their counterparts raised in a barren environment. This work underscores the presence of developmental phenotypic plasticity in cognition among teleost fish, extending its influence to the very earliest stages of an individual's life.

Quantity as a Fish Views It: Behavior and Neurobiology.

An ability to estimate quantities, such as the number of conspecifics or the size of a predator, has been reported in vertebrates. Fish, in particular zebrafish, may be instrumental in advancing the understanding of magnitude cognition. We review here the behavioral studies that have described the ecological relevance of quantity estimation in fish and the current status of the research aimed at investigating the neurobiological bases of these abilities. By combining behavioral methods with molecular genetics and calcium imaging, the involvement of the retina and the optic tectum has been documented for the estimation of continuous quantities in the larval and adult zebrafish brain, and the contributions of the thalamus and the dorsal-central pallium for discrete magnitude estimation in the adult zebrafish brain. Evidence for basic circuitry can now be complemented and extended to research that make use of transgenic lines to deepen our understanding of quantity cognition at genetic and molecular levels.

Cognitive Phenotypic Plasticity: Environmental Enrichment Affects Learning but Not Executive Functions in a Teleost Fish, Poecilia reticulata.

Many aspects of animal cognition are plastically adjusted in response to the environment through individual experience. A remarkable example of this cognitive phenotypic plasticity is often observed when comparing individuals raised in a barren environment to individuals raised in an enriched environment. Evidence of enrichment-driven cognitive plasticity in teleost fish continues to grow, but it remains restricted to a few cognitive traits. The purpose of this study was to investigate how environmental enrichment affects multiple cognitive traits (learning, cognitive flexibility, and inhibitory control) in the guppy, Poecilia reticulata. To reach this goal, we exposed new-born guppies to different treatments: an enrichment environment with social companions, natural substrate, vegetation, and live prey or a barren environment with none of the above. After a month of treatment, we tested the subjects in a battery of three cognitive tasks. Guppies from the enriched environment learned a color discrimination faster compared to guppies from the environment with no enrichments. We observed no difference between guppies of the two treatments in the cognitive flexibility task, requiring selection of a previously unrewarded stimulus, nor in the inhibitory control task, requiring the inhibition of the attack response toward live prey. Overall, the results indicated that environmental enrichment had an influence on guppies' learning ability, but not on the remaining cognitive functions investigated.

Stimulus characteristics, learning bias and visual discrimination in zebrafish (Danio rerio).

Zebrafish is an emerging model in the study of brain function; however, knowledge about its behaviour and cognition is incomplete. Previous studies suggest this species has limited ability in visual learning tasks compared to other teleosts. In this study, we systematically examined zebrafish's ability to learn to discriminate colour, shape, size, and orientation of figures using an appetitive conditioning paradigm. Contrary to earlier reports, the zebrafish successfully completed all tasks. Not all discriminations were learned with the same speed and accuracy. Subjects discriminated the size of objects better than their shape or colour. In all three tasks, they were faster and more accurate when required to discriminate between outlined figures than between filled figures. With stimuli consisting of outlines, the learning performance of zebrafish was comparable to that observed in higher vertebrates. Zebrafish easily learned a horizontal-vertical discrimination task, but like many other vertebrates, they had great difficulty discriminating a figure from its mirror image. Performance was more accurate for subjects reinforced on one stimulus (green over red, triangle over circle, large over small). Unexpectedly, these stimulus biases occurred only when zebrafish were tested with filled figures, suggesting some causal relationship between stimulus preference, learning bias and performance.