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.

Visual discrimination and amodal completion in zebrafish.

While zebrafish represent an important model for the study of the visual system, visual perception in this species is still less investigated than in other teleost fish. In this work, we validated for zebrafish two versions of a visual discrimination learning task, which is based on the motivation to reach food and companions. Using this task, we investigated zebrafish ability to discriminate between two different shape pairs (i.e., disk vs. cross and full vs. amputated disk). Once zebrafish were successfully trained to discriminate a full from an amputated disk, we also tested their ability to visually complete partially occluded objects (amodal completion). After training, animals were presented with two amputated disks. In these test stimuli, another shape was either exactly juxtaposed or only placed close to the missing sectors of the disk. Only the former stimulus should elicit amodal completion. In human observers, this stimulus causes the impression that the other shape is occluding the missing sector of the disk, which is thus perceived as a complete, although partially hidden, disk. In line with our predictions, fish reinforced on the full disk chose the stimulus eliciting amodal completion, while fish reinforced on the amputated disk chose the other stimulus. This represents the first demonstration of amodal completion perception in zebrafish. Moreover, our results also indicated that a specific shape pair (disk vs. cross) might be particularly difficult to discriminate for this species, confirming previous reports obtained with different procedures.

Characterizing ontogeny of quantity discrimination in zebrafish.

A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here, we investigated the ontogeny of this ability using a group size preference (GSP) task in juvenile zebrafish. Fish showed GSP from 21 days post-fertilization and reliably chose the larger group when presented with discriminations of between 1 versus 3, 2 versus 5 and 2 versus 3 conspecifics but not 2 versus 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1 : 2, fish could discriminate between 1 versus 2 individuals and 3 versus 6, but again, not when given a choice between 2 versus 4 individuals. These findings are in agreement with studies in other species, suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fishes may be the result of an interplay between attentional, cognitive and memory-related mechanisms as in humans and other animals. Our results emphasize the potential of the use of zebrafish to explore the genetic and neural processes underlying the ontogeny and function of number cognition.

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.

Learning by Doing: The Use of Distance, Corners and Length in Rewarded Geometric Tasks by Zebrafish (Danio rerio).

Zebrafish spontaneously use distance and directional relationships among three-dimensional extended surfaces to reorient within a rectangular arena. However, they fail to take advantage of either an array of freestanding corners or an array of unequal-length surfaces to search for a no-longer-present goal under a spontaneous cued memory procedure, being unable to use the information supplied by corners and length without some kind of rewarded training. The present study aimed to tease apart the geometric components characterizing a rectangular enclosure under a procedure recruiting the reference memory, thus training zebrafish in fragmented layouts that provided differences in surface distance, corners, and length. Results showed that fish, besides the distance, easily learned to use both corners and length if subjected to a rewarded exit task over time, suggesting that they can represent all the geometrically informative parts of a rectangular arena when consistently exposed to them. Altogether, these findings highlight crucially important issues apropos the employment of different behavioral protocols (spontaneous choice versus training over time) to assess spatial abilities of zebrafish, further paving the way to deepen the role of visual and nonvisual encodings of isolated geometric components in relation to macrostructural boundaries.

The Challenge of Illusory Perception of Animals: The Impact of Methodological Variability in Cross-Species Investigation.

Although we live on the same planet, there are countless different ways of seeing the surroundings that reflect the different individual experiences and selective pressures. In recent decades, visual illusions have been used in behavioural research to compare the perception between different vertebrate species. The studies conducted so far have provided contradictory results, suggesting that the underlying perceptual mechanisms may differ across species. Besides the differentiation of the perceptual mechanisms, another explanation could be taken into account. Indeed, the different studies often used different methodologies that could have potentially introduced confounding factors. In fact, the possibility exists that the illusory perception is influenced by the different methodologies and the test design. Almost every study of this research field has been conducted in laboratories adopting two different methodological approaches: a spontaneous choice test or a training procedure. In the spontaneous choice test, a subject is presented with biologically relevant stimuli in an illusory context, whereas, in the training procedure, a subject has to undergo an extensive training during which neutral stimuli are associated with a biologically relevant reward. Here, we review the literature on this topic, highlighting both the relevance and the potential weaknesses of the different methodological approaches.

Whole brain functional recordings at cellular resolution in zebrafish larvae with 3D scanning multiphoton microscopy.

Optical recordings of neuronal activity at cellular resolution represent an invaluable tool to investigate brain mechanisms. Zebrafish larvae is one of the few model organisms where, using fluorescence-based reporters of the cell activity, it is possible to optically reconstruct the neuronal dynamics across the whole brain. Typically, leveraging the reduced light scattering, methods like lightsheet, structured illumination, and light-field microscopy use spatially extended excitation profiles to detect in parallel activity signals from multiple cells. Here, we present an alternative design for whole brain imaging based on sequential 3D point-scanning excitation. Our approach relies on a multiphoton microscope integrating an electrically tunable lens. We first apply our approach, adopting the GCaMP6s activity reporter, to detect functional responses from retinal ganglion cells (RGC) arborization fields at different depths within the zebrafish larva midbrain. Then, in larvae expressing a nuclear localized GCaMP6s, we recorded whole brain activity with cellular resolution. Adopting a semi-automatic cell segmentation, this allowed reconstructing the activity from up to 52,000 individual neurons across the brain. In conclusion, this design can easily retrofit existing imaging systems and represents a compact, versatile and reliable tool to investigate neuronal activity across the larva brain at high resolution.

Stress reactivity elicits a tissue-specific reduction in telomere length in aging zebrafish (Danio rerio).

Individual differences in personality are associated with variation in healthy aging. Health behaviours are often cited as the likely explanation for this association; however, an underlying biological mechanism may also exist. Accelerated leukocyte telomere shortening is implicated in multiple age-related diseases and is associated with chronic activation of the hypothalamus-pituitary-adrenal (HPA) axis, providing a link between stress-related personality differences and adverse health outcomes. However, the effects of the HPA axis are tissue specific. Thus, leukocyte telomere length may not accurately reflect telomere length in disease-relevant tissues. Here, we examined the correlation between stress reactivity and telomere length in heart and brain tissue in young (6-9 month) and aging (18 month) zebrafish. Stress reactivity was assessed by tank diving and through gene expression. Telomere length was assessed using quantitative PCR. We show that aging zebrafish have shorter telomeres in both heart and brain. Telomere length was inversely related to stress reactivity in heart but not brain of aging individuals. These data support the hypotheses that an anxious predisposition contributes to accelerated telomere shortening in heart tissue, which may have important implications for our understanding of age-related heart disease, and that stress reactivity contributes to age-related telomere shortening in a tissue-specific manner.

Moderate early life stress improves adult zebrafish (Danio rerio) working memory but does not affect social and anxiety-like responses.

Early life stress (ELS) is defined as a short or chronic period of trauma, environmental or social deprivation, which can affect different neurochemical and behavioral patterns during adulthood. Zebrafish (Danio rerio) have been widely used as a model system to understand human neurodevelopmental disorders and display translationally relevant behavioral and stress-regulating systems. In this study, we aimed to investigate the effects of moderate ELS by exposing young animals (6-weeks postfertilization), for 3 consecutive days, to three stressors, and analyzing the impact of this on adult zebrafish behavior (16-week postfertilization). The ELS impact in adults was assessed through analysis of performance on tests of unconditioned memory (free movement pattern Y-maze test), exploratory and anxiety-related task (novel tank diving test), and social cohesion (shoaling test). Here, we show for the first time that moderate ELS increases the number of alternations in turn-direction compared to repetitions in the unconditioned Y-maze task, suggesting increased working memory, but has no effect on shoal cohesion, locomotor profile, or anxiety-like behavior. Overall, our data suggest that moderate ELS may be linked to adaptive flexibility which contributes to build "resilience" in adult zebrafish by improving working memory performance.

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.

Red-footed tortoises (Chelonoidis carbonaria) do not perceive the Delboeuf illusion.

Visual illusions have been widely used as a tool to study animal visual perception. In many cases, identical experimental procedures were adopted to make highly controlled interspecific comparisons. However, reducing methodological variability may prevent reliable comparisons because a certain methodology could be more suitable for some species than others. This study sought to build on previous work that investigated the perception of the Delboeuf illusion in reptiles. Reptiles were presented with trials composed of 2 different-sized food portions on 2 identical plates in which they were expected to maximize their food intake. In contrast to the bearded dragons (Pogona vitticeps), tortoises (Chelonoidis carbonaria) performed poorly in all conditions and therefore no firm conclusion regarding their perception of the illusion could be made. Such results could be due to cognitive challenges or due to the experimental setup, because descending a ramp is demanding for the tortoises. In this study, we adopted the same experimental paradigm but in a flat apparatus. Tortoises significantly discriminated the larger food portions in baseline trials, however, their performance did not differ from chance in illusory trials revealing that, under these conditions, they are not sensitive to the Delboeuf illusion. This nonperception could be ascribed to different factors, such as poorer discrimination ability or a low sensitivity to contrast and assimilation phenomena. Our study highlights the importance of additional investigation to better understand the nature of null results, taking in consideration the ecological needs of the species before drawing any conclusions about its abilities. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

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.

Application of an abstract concept across magnitude dimensions by fish.

Mastering relational concepts and applying them to different contexts presupposes abstraction capacities and implies a high level of cognitive sophistication. One way to investigate extrapolative abilities is to assess cross-dimensional application of an abstract relational magnitude rule to new domains. Here we show that angelfish initially trained to choose either the shorter of two lines in a spatial task (line-length discrimination task) or the array with "fewer" items (numerical discrimination task) spontaneously transferred the learnt rule to novel stimuli belonging to the previously unseen dimension demonstrating knowledge of the abstract concept of "smaller". Our finding challenges the idea that the ability to master abstract magnitude concepts across domains is unique to humans and suggests that the circuits involved in rule learning and magnitude processing might be evolutionary conserved.

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.].

The ontogeny of continuous quantity discrimination in zebrafish larvae (Danio rerio).

Several studies have investigated the ontogeny of the capacity to discriminate between discrete numerical information in human and non-human animals. Contrarily, less attention has been devoted to the development of the capacity to discriminate continuous quantities. Recently, we set up a fast procedure for screening continuous quantity abilities in adult individuals of an animal model in neurodevelopmental research, the zebrafish. Two different sized holes are presented in a wall that divides the home tank in two halves and the spontaneous preference of fish for passing through the larger hole is exploited to measure their discrimination ability. We tested zebrafish larvae in the first, second and third week of life varying the relative size of the smaller circle (0.60, 0.75, 0.86, 0.91 area ratio). We found that the number of passages increased across the age. The capacity to discriminate the larger hole decreased as the ratio between the areas increased. No difference in accuracy was found as a function of age. The accuracy of larval zebrafish almost overlaps that found in adults in a previous study, suggesting a limited role of maturation and experience on the ability to estimate areas in this species.

Exploring the Müller-Lyer illusion in a nonavian reptile (Pogona vitticeps).

Visual illusions have been widely used to compare visual perception among birds and mammals to assess whether animals interpret and alter visual inputs like humans, or if they detect them with little or no variability. Here, we investigated whether a nonavian reptile (Pogona vitticeps) perceives the Müller-Lyer illusion, an illusion that causes a misperception of the relative length of 2 line segments. We observed the animals' spontaneous tendency to choose the larger food quantity (the longer line). In test trials, animals received the same food quantity presented in a spatial arrangement eliciting the size illusion in humans; control trials presented them with 2 different-sized food portions. Bearded dragons significantly selected the larger food quantity in control trials, confirming that they maximized food intake. Group analysis revealed that in the illusory test trials, they preferentially selected the line length estimated as longer by human observers. Further control trials excluded the possibility that their choice was based on potential spatial bias related to the illusory pattern. Our study suggests that a nonavian reptile species has the capability to be sensitive to the Müller-Lyer illusion, raising the intriguing possibility that the perceptual mechanisms underlying size estimation might be similar across amniotes. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Brain and Behavioral Asymmetry: A Lesson From Fish.

It is widely acknowledged that the left and right hemispheres of human brains display both anatomical and functional asymmetries. For more than a century, brain and behavioral lateralization have been considered a uniquely human feature linked to language and handedness. However, over the past decades this idea has been challenged by an increasing number of studies describing structural asymmetries and lateralized behaviors in non-human species extending from primates to fish. Evidence suggesting that a similar pattern of brain lateralization occurs in all vertebrates, humans included, has allowed the emergence of different model systems to investigate the development of brain asymmetries and their impact on behavior. Among animal models, fish have contributed much to the research on lateralization as several fish species exhibit lateralized behaviors. For instance, behavioral studies have shown that the advantages of having an asymmetric brain, such as the ability of simultaneously processing different information and perform parallel tasks compensate the potential costs associated with poor integration of information between the two hemispheres thus helping to better understand the possible evolutionary significance of lateralization. However, these studies inferred how the two sides of the brains are differentially specialized by measuring the differences in the behavioral responses but did not allow to directly investigate the relation between anatomical and functional asymmetries. With respect to this issue, in recent years zebrafish has become a powerful model to address lateralization at different level of complexity, from genes to neural circuitry and behavior. The possibility of combining genetic manipulation of brain asymmetries with cutting-edge in vivo imaging technique and behavioral tests makes the zebrafish a valuable model to investigate the phylogeny and ontogeny of brain lateralization and its relevance for normal brain function and behavior.

Anisotropy of perceived space in non-primates? The horizontal-vertical illusion in bearded dragons (Pogona vitticeps) and red-footed tortoises (Chelonoidis carbonaria).

The horizontal-vertical illusion is a size illusion in which two same-sized objects appear to be different if presented on a horizontal or vertical plane, with the vertical one appearing longer. This illusion represents one of the main evidences of the anisotropy of the perceived space of humans, an asymmetrical perception of the object size presented in the vertical and horizontal space. Although this illusion has been widely investigated in humans, there is an almost complete lack of studies in non-human animals. Here we investigated whether reptiles perceive the horizontal-vertical illusion. We tested two reptile species: bearded dragons (Pogona vitticeps) and red-footed tortoises (Chelonoidis carbonaria). In control trials, two different-sized food strips were presented and animals were expected to choose the longer one. In test trials, animals received two same-sized strips, presented in a spatial arrangement eliciting the illusion. Only bearded dragons significantly preferred the longer strip in control trials; in test trials, bearded dragons selected the strip arranged vertically, suggesting a human-like perception of this pattern, while no clear choice for either array was observed in tortoises. Our results raise the interesting possibility that the anisotropy of perceived space can exists also in a reptile brain.

Food quantity discrimination in puppies (Canis lupus familiaris).

There is considerable evidence that animals are able to discriminate between quantities. Despite the fact that quantitative skills have been extensively studied in adult individuals, research on their development in early life is restricted to a limited number of species. We, therefore, investigated whether 2-month-old puppies could spontaneously discriminate between different quantities of food items. We used a simultaneous two-choice task in which puppies were presented with three numerical combinations of pieces of food (1 vs. 8, 1 vs. 6 and 1 vs. 4), and they were allowed to select only one option. The subjects chose the larger of the two quantities in the 1 vs. 8 and the 1 vs. 6 combinations but not in the 1 vs. 4 combination. Furthermore, the last quantity the puppies looked at before making their choice and the time spent looking at the larger/smaller amounts of food were predictive of the choices they made. Since adult dogs are capable of discriminating between more difficult numerical contrasts when tested with similar tasks, our findings suggest that the capacity to discriminate between quantities is already present at an early age, but that it is limited to very easy discriminations.