Responses in the left and right entopallium are differently affected by light stimulation in embryo.

Sensory stimulation during the prenatal period has been argued to be a main factor in establishing asymmetry in the vertebrate brain. However, though largely studied in behavior and neuroanatomy, nothing is known on the effects of light stimulation in embryo on the activities of single neurons. We performed single-unit recordings from the left and right entopallium of dark- and light-incubated chicks, following ipsi-, contra-, and bilateral visual stimulation. Light incubation increased the general responsiveness of visual neurons in both the left and the right entopallium. Entopallial responses were clearly lateralized in dark-incubated chicks, which showed a general right-hemispheric dominance. This could be suppressed or inverted after light incubation, revealing the presence of both spontaneous and light-dependent asymmetries. These results suggest that asymmetry in single-neuron activity is present at the onset and can be modulated by environmental stimuli such as light exposure in embryos.

Hierarchical processing of feature, egocentric and relational information for spatial orientation in domestic chicks.

Animals can use different types of information for navigation. Domestic chicks (Gallus gallus) prefer to use local features as a beacon over spatial relational information. However, the role of egocentric navigation strategies is less understood. Here, we tested domestic chicks' egocentric and allocentric orientation abilities in a large circular arena. In experiment 1, we investigated whether domestic chicks possess a side bias during viewpoint-dependent egocentric orientation, revealing facilitation for targets on the chicks' left side. Experiment 2 showed that local features are preferred over viewpoint-dependent egocentric information when the two conflict. Lastly, in experiment 3, we found that in a situation where there is a choice between egocentric and allocentric spatial relational information provided by free-standing objects, chicks preferentially rely on egocentric information. We conclude that chicks orient according to a hierarchy of cues, in which the use of the visual appearance of an object is the dominant strategy, followed by viewpoint-dependent egocentric information and finally by spatial relational information.

Naïve chicks do not prefer objects with stable body orientation, though they may prefer behavioural variability.

Domestic chicks (Gallus gallus domesticus) have been widely used as a model to study the motion cues that allow visually naïve organisms to detect animate agents shortly after hatching/birth. Our previous work has shown that chicks prefer to approach agents whose main body axis and motion direction are aligned (a feature typical of creatures whose motion is constrained by a bilaterally symmetric body plan). However, it has never been investigated whether chicks are also sensitive to the fact that an agent maintains a stable front-back body orientation in motion (i.e. consistency in which end is leading and which trailing). This is another feature typical of bilateria, which is also associated with the detection of animate agents in humans. The aim of the present study was to fill this gap. Contrary to our initial expectations, after testing 300 chicks across 3 experimental conditions, we found a recurrent preference for the agent which did not maintain a stable front-back body orientation. Since this preference was limited to female chicks, the results are discussed also in relation to sex differences in the social behaviour of this model. Overall, we show for the first time that chicks can discriminate agents based on the stability of their front-back orientation. The unexpected direction of the effect could reflect a preference for agents' whose behaviour is less predictable. Chicks may prefer agents with greater behavioural variability, a trait which has been associated with animate agents, or have a tendency to explore agents performing "odd behaviours".

Spontaneous preference for unpredictability in the temporal contingencies between agents' motion in naive domestic chicks.

The ability to recognize animate agents based on their motion has been investigated in humans and animals alike. When the movements of multiple objects are interdependent, humans perceive the presence of social interactions and goal-directed behaviours. Here, we investigated how visually naive domestic chicks respond to agents whose motion was reciprocally contingent in space and time (i.e. the time and direction of motion of one object can be predicted from the time and direction of motion of another object). We presented a 'social aggregation' stimulus, in which three smaller discs repeatedly converged towards a bigger disc, moving in a manner resembling a mother hen and chicks (versus a control stimulus lacking such interactions). Remarkably, chicks preferred stimuli in which the timing of the motion of one object could not be predicted by that of other objects. This is the first demonstration of a sensitivity to the temporal relationships between the motion of different objects in naive animals, a trait that could be at the basis of the development of the perception of social interaction and goal-directed behaviours.

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.

Light-incubation effects on lateralisation of single unit responses in the visual Wulst of domestic chicks.

Since the ground-breaking discovery that in-egg light exposure triggers the emergence of visual lateralisation, domestic chicks became a crucial model for research on the interaction of environmental and genetic influences for brain development. In domestic chick embryos, light exposure induces neuroanatomical asymmetries in the strength of visual projections from the thalamus to the visual Wulst. Consequently, the right visual Wulst receives more bilateral information from the two eyes than the left one. How this impacts visual Wulst's physiology is still unknown. This paper investigates the visual response properties of neurons in the left and right Wulst of dark- and light-incubated chicks, studying the effect of light incubation on bilaterally responsive cells that integrate information from both eyes. We recorded from a large number of visually responsive units, providing the first direct evidence of lateralisation in the neural response properties of units of the visual Wulst. While we confirm that some forms of lateralisation are induced by embryonic light exposure, we found also many cases of light-independent asymmetries. Moreover, we found a strong effect of in-egg light exposure on the general development of the functional properties of units in the two hemispheres. This indicates that the effect of embryonic stimulation goes beyond its contribution to the emergence of some forms of lateralisation, with influences on the maturation of visual units in both hemispheres.

Visual Field Analysis: A reliable method to score left and right eye use using automated tracking.

Brain and behavioural asymmetries have been documented in various taxa. Many of these asymmetries involve preferential left and right eye use. However, measuring eye use through manual frame-by-frame analyses from video recordings is laborious and may lead to biases. Recent progress in technology has allowed the development of accurate tracking techniques for measuring animal behaviour. Amongst these techniques, DeepLabCut, a Python-based tracking toolbox using transfer learning with deep neural networks, offers the possibility to track different body parts with unprecedented accuracy. Exploiting the potentialities of DeepLabCut, we developed Visual Field Analysis, an additional open-source application for extracting eye use data. To our knowledge, this is the first application that can automatically quantify left-right preferences in eye use. Here we test the performance of our application in measuring preferential eye use in young domestic chicks. The comparison with manual scoring methods revealed a near perfect correlation in the measures of eye use obtained by Visual Field Analysis. With our application, eye use can be analysed reliably, objectively and at a fine scale in different experimental paradigms.

Spontaneous Visual Preference for Face-Like Stimuli Is Impaired in Newly-Hatched Domestic Chicks Exposed to Valproic Acid During Embryogenesis.

Faces convey a great amount of socially relevant information related to emotional and mental states, identity and intention. Processing of face information is a key mechanism for social and cognitive development, such that newborn babies are already tuned to recognize and orient to faces and simple schematic face-like patterns since the first hours of life. Similar to neonates, also non-human primates and domestic chicks have been shown to express orienting responses to faces and schematic face-like patterns. More importantly, existing studies have hypothesized that early disturbances of these mechanisms represent one of the earliest biomarker of social deficits in autism spectrum disorders (ASD). We used VPA exposure to induce neurodevelopmental changes associated with ASD in domestic chicks and tested whether VPA could impact the expression of the animals' approach responses to schematic face-like stimuli. We found that VPA impairs the chicks' preference responses to these social stimuli. Based on the results shown here and on previous studies, we propose the domestic chick as animal model to investigate the biological mechanisms underlying face processing deficits in ASD.

Abnormal visual attention to simple social stimuli in 4-month-old infants at high risk for Autism.

Despite an increasing interest in detecting early signs of Autism Spectrum Disorders (ASD), the pathogenesis of the social impairments characterizing ASD is still largely unknown. Atypical visual attention to social stimuli is a potential early marker of the social and communicative deficits of ASD. Some authors hypothesized that such impairments are present from birth, leading to a decline in the subsequent typical functioning of the learning-mechanisms. Others suggested that these early deficits emerge during the transition from subcortically to cortically mediated mechanisms, happening around 2-3 months of age. The present study aimed to provide additional evidence on the origin of the early visual attention disturbance that seems to characterize infants at high risk (HR) for ASD. Four visual preference tasks were used to investigate social attention in 4-month-old HR, compared to low-risk (LR) infants of the same age. Visual attention differences between HR and LR infants emerged only for stimuli depicting a direct eye-gaze, compared to an adverted eye-gaze. Specifically, HR infants showed a significant visual preference for the direct eye-gaze stimulus compared to LR infants, which may indicate a delayed development of the visual preferences normally observed at birth in typically developing infants. No other differences were found between groups. Results are discussed in the light of the hypotheses on the origins of early social visual attention impairments in infants at risk for ASD.

Anatomical asymmetries in the tectofugal pathway of dark-incubated domestic chicks: Rightwards lateralization of parvalbumin neurons in the entopallium.

The discovery of the role of light exposure for the development of lateralization in domestic chick embryos revolutionized this research field. However, two main issues remain unresolved: (i) while in chicks anatomical light-dependent lateralization is mostly confined to the thalamofugal visual pathway, in pigeons only the tectofugal pathway is lateralized after light exposure. However, no study in either species ever investigated anatomical lateralization in the entopallium, the forebrain station of the tectofugal pathway. (ii) It is now known that lateralization can be observed also in dark-incubated chicks, both at the behavioural and at the Immediate Early Gene-expression level. We hypothesized that lateralization of the tectofugal system may underlie these light-independent effects. To investigate structural lateralization in the tectofugal pathway of dark-incubated chicks, we used parvalbumin (PV) as a marker of a sub population of entopallial neurons, quantifying PV-ir cell densities in the left and right entopallium. We found higher density in the right hemisphere, revealing for the first time anatomical lateralization in entopallium and confirming its potential role in supporting lateralized brain processing in dark-incubated birds. Results are discussed in relation to the possible functional role of PV-ir cells in inhibitory neural functions.

Sensitive periods for social development: Interactions between predisposed and learned mechanisms.

We analysed research that makes use of precocial species as animal models to describe the interaction of predisposed mechanisms and environmental factors in early learning, in particular for the development of social cognition. We also highlight the role of sensitive periods in this interaction, focusing on domestic chicks as one of the main animal models for this field. In the first section of the review, we focus on the emergence of early predispositions to attend to social partners. These attentional biases appear before any learning experience about social stimuli. However, non-specific experiences occurring during sensitive periods of the early post-natal life determine the emergence of these predisposed mechanisms for the detection of social partners. Social predispositions have an important role for the development learning-based social cognitive functions, showing the interdependence of predisposed and learned mechanisms in shaping social development. In the second part of the review we concentrate on the reciprocal interactions between filial imprinting and spontaneous (not learned) social predispositions. Reciprocal influences between these two sets of mechanisms ensure that, in the natural environment, filial imprinting will target appropriate social objects. Neural and physiological mechanisms regulating the sensitive periods for the emergence of social predispositions and for filial imprinting learning are also described.

Neural basis of unfamiliar conspecific recognition in domestic chicks (Gallus Gallus domesticus).

Domestic chickens are able to distinguish familiar from unfamiliar conspecifics, however the neuronal mechanisms mediating this behaviour are almost unknown. Moreover, the lateralisation of chicks' social recognition has only been investigated at the behavioural level, but not at the neural level. The aim of the present study was to test the hypothesis that exposure to unfamiliar conspecifics will selectively activate septum, hippocampus or nucleus taeniae of the amygdala of young domestic chicks. Moreover we also wanted to test the lateralisation of this response. For this purpose, we used the immediate early gene product c-Fos to map neural activity. Chicks were housed in pairs for one week. At test, either one of the two chicks was exchanged by an unfamiliar individual (experimental 'unfamiliar' group) or the familiar individual was briefly removed and then placed back in its original cage (control 'familiar' group). Analyses of chicks' interactions with the familiar/unfamiliar social companion revealed a higher number of social pecks directed towards unfamiliar individuals, compared to familiar controls. Moreover, in the group exposed to the unfamiliar individual a significantly higher activation was present in the dorsal and ventral septum of the left hemisphere and in the ventral hippocampus of the right hemisphere, compared to the control condition. These effects were neither present in other subareas of hippocampus or septum, nor in the nucleus taeniae of the amygdala. Our study thus indicates selective lateralised involvement of domestic chicks' septal and hippocampal subregions in responses to unfamiliar conspecific.

Statistical learning in domestic chicks is modulated by strain and sex.

Statistical learning is a key mechanism for detecting regularities from a variety of sensory inputs. Precocial newborn domestic chicks provide an excellent model for (1) exploring unsupervised forms of statistical learning in a comparative perspective, and (2) elucidating the ecological function of statistical learning using imprinting procedures. Here we investigated the role of the sex of the chicks in modulating the direction of preference (for familiarity or novelty) in a visual statistical learning task already employed with chicks and human infants. Using both automated tracking and direct human coding, we confirmed chicks' capacity to recognize the presence of a statistically defined structure underlying a continuous stream of shapes. Using a different chicken strain than previous studies, we were also able to highlight sex differences in chicks' propensity to approach the familiar or novel sequence. This could also explain a previous failure to reveal statistical learning in chicks which sex was however not determined. Our study confirms chicks' ability to track visual statistics. The pivotal role of sex in determining familiarity or novelty preferences in this species and the interaction with the animals' strain highlight the importance to contextualize comparative research within the ecology of each species.

Unlearned visual preferences for the head region in domestic chicks.

Unlearned tendencies to approach animate creatures are of great adaptive value, especially for nidifugous social birds that need to react to the presence of potential social companions shortly after hatching. Domestic chicks' preferences for taxidermized hens provided the first evidence of social predispositions. However, the nature of the stimuli eliciting this predisposition is not completely understood. Here we explore the unlearned preferences of visually naïve domestic chicks for taxidermized animals. Visually naive chicks were tested for their approach preferences between a target stimulus (an intact stuffed animal whose head region was clearly visible) and a control stimulus. After confirming the predisposition for the intact stuffed fowl hen (Exp. 1), we found an analogous preference for a taxidermized, young domestic chick over a severely scrambled version of the same stimulus, whose body structure was completely disrupted, extending to same-age individuals the results that had been obtained with taxidermized hens (Exp. 2). We also directly tested preferences for specimens whose head region is visible compared to ones whose head region was occluded. To clarify whether chicks are sensitive to species-specific information, we employed specimens of female mallard ducks and of a mammalian predator, the polecat. Chicks showed a preference for the duck stimulus whose wings have been covered over a similar stimulus whose head region has been covered, providing direct evidence that the visibility of the head region of taxidermized models drive chicks' behaviour in this test, and that the attraction for the head region indeed extends to females of other bird species (Exp. 3). However, no similar preference was obtained with the polecat stimuli (Exp. 4). We thus confirmed the presence of unlearned visual preferences for the head region in newly-hatched chicks, though other factors can limit the species-generality of the phenomenon.

Selective response of the nucleus taeniae of the amygdala to a naturalistic social stimulus in visually naive domestic chicks.

The detection of animate beings at the onset of life is important for phylogenetically distant species, such as birds and primates. Naïve chicks preferentially approach a stimulus resembling a conspecific (a stuffed fowl) over a less naturalistic one (a scrambled version of the stuffed fowl, presenting the same low-level visual features as the fowl in an unnatural configuration). The neuronal mechanisms underlying this behavior are mostly unknown. However, it has been hypothesized that innate social predispositions may involve subpallial brain areas including the amygdala. Here we asked whether a stuffed hen would activate areas of the arcopallium/amygdala complex, in particular the nucleus taeniae of the amygdala (TnA) or septum. We measured brain activity by visualizing the immediate early gene product c-Fos. After exposure to the hen, TnA showed higher density of c-Fos expressing neurons, compared to chicks that were exposed to the scrambled stimulus. A similar trend was present in the lower portion of the arcopallium, but not in the upper portion of the arcopallium or in the septum. This demonstrates that at birth the TnA is already engaged in responses to social visual stimuli, suggesting an important role for this nucleus in the early ontogenetic development of social behavior.

Embryonic Exposure to Valproic Acid Affects Social Predispositions for Dynamic Cues of Animate Motion in Newly-Hatched Chicks.

Early predispositions to preferentially orient toward cues associated with social partners have been documented in several vertebrate species including human neonates and domestic chicks. Human newborns at high familiar risk of Autism Spectrum Disorder (ASD) show differences in their attention toward these predisposed stimuli, suggesting potential impairments in the social-orienting mechanisms in ASD. Using embryonic exposure to valproic acid (VPA) we modeled ASD behavioral deficits in domestic chicks. To investigate social predispositions toward animate motion in domestic chicks, we focused on self-propulsion, using two video-animations representing a simple red circle moving at constant speed (speed-constant) or one that was changing its speed (accelerating and decelerating; speed-change). Using a spontaneous choice test for the two stimuli, we compared spontaneous preferences for stimuli that autonomously change speed between VPA- and vehicle-injected chicks. We found that the preference for speed changes was abolished in VPA-injected chicks compared to vehicle-injected controls. These results add to previous findings indicating similar impairments for static social stimuli and suggest a specific effect of VPA on the development of mechanisms that enhance orienting toward animate stimuli. These findings strengthen the hypothesis of an early impairment of predispositions in the early development of ASD. Hence, early predispositions are a potentially useful tool to detect early ASD symptoms in human neonates and to investigate the molecular and neurobiological mechanisms underlying the onset of this neurodevelopmental disorder.

Spontaneous and light-induced lateralization of immediate early genes expression in domestic chicks.

Exposure of domestic chicks' eggs to light during embryo incubation stimulates asymmetrically the two eye-systems, reaching selectively the right eye (left hemisphere) and inducing asymmetries at the behavioral and neural level. Surprisingly, though, some types of lateralization have been observed also in dark incubated chicks, especially at the behavioral level. Here we investigate the mechanisms subtending the development of lateralization, in the presence and in the absence of embryonic light exposure. We measured the baseline level of expression for the immediate early gene product c-Fos, used as an indicator of the spontaneous level of neural activity and plasticity in four areas of the two hemispheres (preoptic area, septum, hippocampus and intermediate medial mesopallium). Additional DAPI staining measured overall cell density (regardless of c-Fos expression), ruling out any confound due to underlying asymmetries in cell density between the hemispheres. In different brain areas, c-Fos expression was lateralized either in light- (septum) or in dark-incubated chicks (preoptic area). Light exposure increased c-Fos expression in the left hemisphere, suggesting that c-Fos expression could participate to the known effects of light stimulation on brain asymmetries. Interestingly, this effect was visible few days after the end of the light exposure, revealing a delayed effect of light exposure on c-Fos baseline expression in brain areas outside the visual pathways. In the preoptic area of dark incubated chicks, we found a rightward bias for c-Fos expression, revealing that lateralization of the baseline level of activity and plasticity is present in the developing brain also in the absence of light exposure.

Spontaneous Learning of Visual Structures in Domestic Chicks.

Effective communication crucially depends on the ability to produce and recognize structured signals, as apparent in language and birdsong. Although it is not clear to what extent similar syntactic-like abilities can be identified in other animals, recently we reported that domestic chicks can learn abstract visual patterns and the statistical structure defined by a temporal sequence of visual shapes. However, little is known about chicks' ability to process spatial/positional information from visual configurations. Here, we used filial imprinting as an unsupervised learning mechanism to study spontaneous encoding of the structure of a configuration of different shapes. After being exposed to a triplet of shapes (ABC or CAB), chicks could discriminate those triplets from a permutation of the same shapes in different order (CAB or ABC), revealing a sensitivity to the spatial arrangement of the elements. When tested with a fragment taken from the imprinting triplet that followed the familiar adjacency-relationships (AB or BC) vs. one in which the shapes maintained their position with respect to the stimulus edges (AC), chicks revealed a preference for the configuration with familiar edge elements, showing an edge bias previously found only with temporal sequences.

Embryonic Exposure to Valproic Acid Impairs Social Predispositions of Newly-Hatched Chicks.

Biological predispositions to attend to visual cues, such as those associated with face-like stimuli or with biological motion, guide social behavior from the first moments of life and have been documented in human neonates, infant monkeys and domestic chicks. Impairments of social predispositions have been recently reported in neonates at high familial risk of Autism Spectrum Disorder (ASD). Using embryonic exposure to valproic acid (VPA), an anticonvulsant associated to increased risk of developing ASD, we modeled ASD behavioral deficits in domestic chicks. We then assessed their spontaneous social predispositions by comparing approach responses to a stimulus containing a face configuration, a stuffed hen, vs. a scrambled version of it. We found that this social predisposition was abolished in VPA-treated chicks, whereas experience-dependent mechanisms associated with filial imprinting were not affected. Our results suggest a specific effect of VPA on the development of biologically-predisposed social orienting mechanisms, opening new perspectives to investigate the neurobiological mechanisms involved in early ASD symptoms.

Dynamic features of animate motion activate septal and preoptic areas in visually naïve chicks (Gallus gallus).

The septum is an evolutionarily well-conserved part of the limbic system. It is known to be involved in many aspects of social behavior and is considered a key node of the social behavior network, together with the preoptic area. Involvement of these two brain regions has been recently observed in newly hatched chicks exposed to the natural motion of a living conspecific. However, it is unknown whether these areas respond also to simple motion cues that elicit animacy perception in humans and social predispositions in chicks. For example, naïve chicks are attracted by visual objects that appear to spontaneously change their speed (an index of self-propulsion, typical of animate creatures). Here we show that the right septum and the preoptic area of newly hatched visually naïve chicks exposed to speed changes have higher neuronal activity (revealed by c-Fos expression), compared with that of chicks exposed to constant motion. We thus found an involvement of these two areas in the perception of motion cues associated with animacy in newly hatched chicks without any previous visual experience. This demonstrates their early involvement in processing simple motion cues that allow the detection of animate creatures and elicit social predispositions in this animal model, as well as preferential attention in human infants and the perception of animacy in human adults.