Exploring the Importance of Environmental Complexity for Newly Hatched Zebrafish.

The effects of an early impoverished social or physical environment on vertebrate neural development and cognition has been known for decades. While existing studies have focused on the long-term effects, measuring adult cognitive phenotypes, studies on the effects of environmental complexity on the early stages of development are lacking. Zebrafish (Danio rerio) hatchlings are assumed to have minimal interaction with their environment and are routinely reared in small, bare containers. To investigate the effects of being raised under such conditions on development of behaviour and cognition, hatchlings housed for 10 days in either an enriched or a standard environment underwent two cognitive tasks. The results were mixed. Subjects of the two treatments did not differ in performance when required to discriminate two areas. Conversely, we found a significant effect in a number discrimination task, with subjects from impoverished condition performing significantly worse. In both experiments, larvae reared in impoverished environment showed a reduced locomotor activity. Given the effects that enrichment appears to exert on larvae, a third experiment explored whether hatchlings exhibit a spontaneous preference for more complex environments. When offered a choice between a bare setting and one with objects of different shapes and colors, larvae spent over 70% of time in the enriched sector. Deepening these effects of an early impoverished environment on cognitive development is crucial for the welfare of captive zebrafish populations and for enhancing the quality and reliability of studies on larval zebrafish.

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.

Shortest path choice in zebrafish (Danio rerio).

Many animals regularly move between different locations within their home range. During these journeys, individuals are expected to use the shortest path, because this strategy minimizes energy expenditure and reduces exposure to adverse conditions, such as predation. The ability to find the shortest distance route has been demonstrated in ants, migrating birds and a few mammals. We investigated whether a freshwater fish, Danio rerio, exhibits this ability. Small groups of zebrafish were allowed to move between the two compartments of their tank using two paths differing in length. They developed a preference for the shorter path gradually over the six days of the experiment. Subjects' accuracy in choosing the shorter path varied from below 60%, with a 20% length disparity, to 80% when one path was twice as long as the other. In a second experiment, zebrafish were initially allowed to practice in groups and then tested individually. We found evidence of individual and sex differences in performance, with males performing more accurately than females. However, due to our experimental design, we cannot conclusively determine whether these differences are indeed cognitive or influenced by confounding factors during the group phase of the experiment.

Illu-Shoal Choice: An Exploration of Different Means for Enrichment of Captive Zebrafish.

Fish of any variety are nowadays being kept captive for several purposes, from recreational to alimentary to research. It is possible that we humans often underestimate or misunderstand the basic, natural needs of the species we use for our purposes. Sociality is likely to play an extensive and fundamental role in the quality of life of animals such as zebrafish. This study aimed to develop a dummy conspecific that included depth and motion illusions in order to assess whether these stimuli could represent a valid alternative to a conspecific in triggering shoaling behaviour in a well-known model in genetics and neuroscience, the zebrafish (Dario rerio). We thus replaced the natural livery of a zebrafish shape with three visual illusions: the Ouchi-Spillmann illusion, which generates an effect of local tilting motion; and another two which should create pictorial cues of tridimensionality. Via a binary shoal choice test, we assessed the time spent close to each of the three artificial dummies compared to neutral control stimuli such as grey ellipses. We found no preference for the illusory patterns, suggesting that the illusion was not perceived or, alternatively, that the perception of the illusion was not enough to elicit recognition of the dummy as conspecific and subsequent social behaviours.

Learning and visual discrimination in newly hatched zebrafish.

With the exception of humans, early cognitive development has been thoroughly investigated only in precocial species, well developed at birth and with a broad behavioral and cognitive repertoire. We investigated another highly altricial species, the zebrafish, Danio rerio, whose embryonic development is very rapid (< 72 h). The hatchlings' nervous system is poorly developed, and their cognitive capacities are largely unknown. Larvae trained at 8 days post fertilization rapidly learned to associate a visual pattern with a food reward, showing significant performance at 10 days post fertilization. We exploited this ability to study hatchlings' discrimination learning capacities. Larvae rapidly and accurately learned color and shape discriminations. They also discriminated a figure from its mirror image and from its 90°-rotated version, although with lower performance. Our study revealed impressive similarities in learning and visual discrimination capacities between newborn and adult zebrafish, despite their enormous differences in brain size and degree of development.

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.

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.

Automated Operant Conditioning Devices for Fish. Do They Work?

The growing use of teleosts in comparative cognition and in neurobiological research has prompted many researchers to develop automated conditioning devices for fish. These techniques can make research less expensive and fully comparable with research on warm-blooded species, in which automated devices have been used for more than a century. Tested with a recently developed automated device, guppies (Poecilia reticulata) easily performed 80 reinforced trials per session, exceeding 80% accuracy in color or shape discrimination tasks after only 3-4 training session, though they exhibit unexpectedly poor performance in numerical discrimination tasks. As several pieces of evidence indicate, guppies possess excellent numerical abilities. In the first part of this study, we benchmarked the automated training device with a standard manual training procedure by administering the same set of tasks, which consisted of numerical discriminations of increasing difficulty. All manually-trained guppies quickly learned the easiest discriminations and a substantial percentage learned the more difficult ones, such as 4 vs. 5 items. No fish trained with the automated conditioning device reached the learning criterion for even the easiest discriminations. In the second part of the study, we introduced a series of modifications to the conditioning chamber and to the procedure in an attempt to improve its efficiency. Increasing the decision time, inter-trial interval, or visibility of the stimuli did not produce an appreciable improvement. Reducing the cognitive load of the task by training subjects first to use the device with shape and color discriminations, significantly improved their numerical performance. Allowing the subjects to reside in the test chamber, which likely reduced the amount of attentional resources subtracted to task execution, also led to an improvement, although in no case did subjects match the performance of fish trained with the standard procedure. Our results highlight limitations in the capacity of small laboratory teleosts to cope with operant conditioning automation that was not observed in laboratory mammals and birds and that currently prevent an easy and straightforward comparison with other vertebrates.

Are cerebral and behavioural lateralization related to anxiety-like traits in the animal model zebrafish (Danio rerio)?

Brain lateralization refers to hemispheric asymmetries in functions and/or neuroanatomical structures. Functional specialization in non-human animals has been mainly inferred through observation of lateralized motor responses and sensory perception. Only in a few cases has the influence of brain asymmetries on behaviour been described. Zebrafish has rapidly become a valuable model to investigate this issue as it displays epithalamic asymmetries that have been correlated to some lateralized behaviours. Here we investigated the relation between neuroanatomical or behavioural lateralization and anxiety using a light-dark preference test in adult zebrafish. In Experiment 1, we observed how scototaxis response varied as a function of behavioural lateralization measured in the detour task as turning preference in front of a dummy predator. In Experiment 2, foxD3:GFP transgenic adult zebrafish with left or right parapineal position, were tested in the same light-dark test as fish in Experiment 1. No correlation was found between the behaviour observed in the detour test and in the scototaxis test nor between the left- and right-parapineal fish and the scototaxis response. The consistency of results obtained in both experiments indicates that neither behavioural nor neuroanatomical asymmetries are related to anxiety-related behaviours measured in the light-dark test.

Forest before the trees in the aquatic world: global and local processing in teleost fishes.

BACKGROUND: The study of illusory phenomena is important to understanding the similarities and differences between mammals and birds' perceptual systems. In recent years, the analysis has been enlarged to include cold-blooded vertebrates, such as fish. However, evidence collected in the literature have drawn a contradictory picture, with some fish species exhibiting a human-like perception of visual illusions and others showing either a reversed perception or no susceptibility to visual illusions. The possibility exists that these mixed results relate to interspecific variability in perceptual grouping mechanisms. Therefore, we studied whether fish of five species exhibit a spontaneous tendency to prioritize a global analysis of the visual scene-also known as global-to-local precedence-instead of focusing on local details. METHODS: Using Navon-like stimuli (i.e., larger recognisable shapes composed of copies of smaller different shapes), we trained redtail splitfin, zebrafish, angelfish, Siamese fighting fish and three spot gourami to discriminate between two figures characterized by congruency between global and local information (a circle made by small circles and a cross made by small crosses). In the test phase, we put global and local cues (e.g., a circle made by small crosses) into contrast to see whether fish spontaneously rely on global or local information. RESULTS: Like humans, fish seem to have an overall global-to-local precedence, with no significant differences among the species. However, looking at the species-specific level, only four out of five species showed a significant global-to-local precedence, and at different degrees. Because these species are distantly related and occupy a broad spectrum of ecological adaptations, we suggest that the tendency to prioritize a global analysis of visual inputs may be more similar in fish than expected by the mixed results of visual illusion studies.

Susceptibility to Size Visual Illusions in a Non-Primate Mammal (Equus caballus).

The perception of different size illusions is believed to be determined by size-scaling mechanisms that lead individuals to extrapolate inappropriate 3D information from 2D stimuli. The Muller-Lyer illusion represents one of the most investigated size illusions. Studies on non-human primates showed a human-like perception of this illusory pattern. To date, it is not clear whether non-primate mammals experience a similar illusory effect. Here, we investigated whether horses perceive the Muller-Lyer illusion by using their spontaneous preference for the larger portion of carrot. In control trials, we presented horses with two carrot sticks of different sizes, and in test trials, carrot sticks of identical size were shown to the subjects together with arrowheads made of plastic material and arranged in a way meant to elicit the Müller-Lyer illusion in human observers. In control trials, horses significantly discriminated between the smaller and larger carrot stick. When presented with the illusion, they showed a significant preference for the carrot that humans perceive as longer. Further control trials excluded the possibility that their choices were based on the total size of the carrot stick and the arrowheads together. The susceptibility of horses to this illusion indicates that the perceptual mechanisms underlying size estimation in perissodactyla might be similar to those of primates, notwithstanding the considerable evolutionary divergence in the visual systems of these two mammalian groups.

Corrigendum: Searching for the Critical p of Macphail's Null Hypothesis: The Contribution of Numerical Abilities of Fish.

[This corrects the article on p. 55 in vol. 11, PMID: 32116895.].

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.

The devil is in the detail: Zebrafish learn to discriminate visual stimuli only if salient.

Due to their unique characteristics, the zebrafish plays a key role in the comprehension of neurobiology of cognition and its pathologies, such as neurodegenerative diseases. More and more molecular tools for this aim are being developed, but our knowledge about the cognitive abilities of zebrafish remains extremely scarce compared to other teleost fish. We aimed to investigate the complex cognitive abilities of zebrafish using a tracking-based automated conditioning chamber that allowed precise experimental control, avoided potential cueing provided by the observer (Clever Hans phenomenon), and was shown to considerably improve learning in other teleosts. A computer presented two visual stimuli in two sectors of the chamber, and zebrafish had to enter the correct sector to obtain a food reward. Zebrafish quickly learned to use the conditioning device and easily performed up to 80 trials per day. In Experiment 1, zebrafish efficiently discriminated between two differently coloured sides, reaching a 75 % accuracy in only 10 training sessions. Surprisingly, zebrafish failed to choose the correct chamber when the stimuli were two shapes, a small circle and a small triangle, even when, in Experiment 2, training on shape discrimination was prolonged for up to 30 sessions. In Experiment 3, we tested the hypothesis that simultaneously learning to use the conditioning chamber and learning discrimination imposes a too-high cognitive load. However, zebrafish that first successfully learned how the conditioning chamber functioned (in the colour discrimination) subsequently failed in the shape discrimination. Conversely, zebrafish that firstly failed the shape discrimination subsequently learned colour discrimination. In Experiment 4, zebrafish showed some evidence of learning when the stimuli were two large shapes, suggesting that zebrafish did not discriminate between the shapes of the previous experiments because they were not salient enough. Altogether, results suggest constraints in the discrimination learning abilities of zebrafish, which should be taken into account when developing cognitive tasks for this species.

Vegetation cover induces developmental plasticity of lateralization in tadpoles.

Lateralization of cognitive functions influences a large number of fitness-related behaviors and shows, in most species, substantial variation in strength and direction. Laboratory works and field data have suggested that this variation is often due to adaptive phenotypic plasticity. Strong lateralization should be favored in some ecological conditions, for example, under high risk of predation. For anuran tadpoles, the presence of cover affects predation risk, with tadpoles being more exposed to predators in environments with reduced cover. We tested the hypothesis that the amount of cover experienced early in life affects lateralization in the edible frog, Pelophylax esculentus, tadpoles. We exposed embryos and larvae to high or low vegetation cover environments. For half of the subjects, the treatment was constant whereas the remaining subjects were switched to the opposite treatment after hatching. In agreement with the theoretical expectation, tadpoles exposed to low vegetation cover for the entire development were more lateralized and showed a stronger alignment in directionality of lateralization compared with tadpoles exposed to high vegetation cover. This indicates a possible role of natural variation in vegetation abundance and developmental plasticity as determinants of between-population and between-individual differences in lateralization. We also found that shifting from high to low vegetation cover treatments and vice versa disrupted lateralization alignment, suggesting that developmental trajectories for this trait are determined at the embryonic stage and need environmental stability to be fully expressed.

Searching for the Critical p of Macphail's Null Hypothesis: The Contribution of Numerical Abilities of Fish.

In 1985, Macphail argued that there are no differences among the intellects of non-human vertebrates and that humans display unique cognitive skills because of language. Mathematical abilities represent one of the most sophisticated cognitive skills. While it is unquestionable that humans exhibit impressive mathematical skills associated with language, a large body of experimental evidence suggests that Macphail hypothesis must be refined in this field. In particular, the evidence that also small-brained organisms, such as fish, are capable of processing numerical information challenges the idea that humans display unique cognitive skills. Like humans, fish may take advantage of using continuous quantities (such as the area occupied by the objects) as proxy of number to select the larger/smaller group. Fish and humans also showed interesting similarities in the strategy adopted to learn a numerical rule. Collective intelligence in numerical estimation has been also observed in humans and guppies. However, numerical acuity in humans is considerably higher than that reported in any fish species investigated, suggesting that quantitative but not qualitative differences do exist between humans and fish. Lastly, while it is clear that contextual factors play an important role in the performance of numerical tasks, inter-species variability can be found also when different fish species were tested in comparable conditions, a fact that does not align with the null hypothesis of vertebrate intelligence. Taken together, we believe that the recent evidence of numerical abilities in fish call for a deeper reflection of Macphail's hypothesis.

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.

A review and consideration on the kinematics of reach-to-grasp movements in macaque monkeys.

The bases for understanding the neuronal mechanisms that underlie the control of reach-to-grasp movements among nonhuman primates, particularly macaques, has been widely studied. However, only a few kinematic descriptions of their prehensile actions are available. A thorough understanding of macaques' prehensile movements is manifestly critical, in light of their role in biomedical research as valuable models for studying neuromotor disorders and brain mechanisms, as well as for developing brain-machine interfaces to facilitate arm control. This article aims to review the current state of knowledge on the kinematics of grasping movements that macaques perform in naturalistic, seminaturalistic, and laboratory settings, to answer the following questions: Are kinematic signatures affected by the context within which the movement is performed? In what ways are kinematics of humans' and macaques' prehensile actions similar/dissimilar? Our analysis reflects the challenges involved in making comparisons across settings and species due to the heterogeneous picture in terms of the number of subjects, stimuli, conditions, and hands used. The kinematics of free-ranging macaques are characterized by distinctive features that are exhibited neither by macaques in laboratory setting nor by human subjects. The temporal incidence of key kinematic landmarks diverges significantly between species, indicating disparities in the overall organization of movement. Given such complexities, we attempt a synthesis of the extant body of evidence, intending to generate some significant implications for directions that future research might take to recognize the remaining gaps and pursue the insights and resolutions to generate an interpretation of movement kinematics that accounts for all settings and subjects.

Personality and Cognition: Sociability Negatively Predicts Shoal Size Discrimination Performance in Guppies.

Evidence from a growing number of organisms suggests that individuals show consistent performance differences in cognitive tasks. According to empirical and theoretical studies, these cognitive differences might be at least partially related to personality. We tested this hypothesis in the guppy, Poecilia reticulata, by comparing individuals with different degree of sociability in the discrimination of shoals formed by a different number of conspecifics. We found that individual guppies show repeatability of sociability as expected for personality traits. Furthermore, individuals with higher sociability showed poorer shoal size discrimination performance and were less efficient in choosing the larger shoal compared to individuals with low sociability. As choosing the larger shoal is an important strategy of defense against predators for guppies, we discuss this relationship between personality and cognition in the light of its fitness consequences.

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.