Functional MRI of imprinting memory: a new avenue for neurobiology of early learning.

Filial imprinting, a crucial ethological paradigm, provides insights into the neurobiology of early learning and its long-term impact on behaviour. To date, only invasive techniques, such as autoradiography or lesion, have been employed to understand this behaviour. The primary limitation of these methods lies in their constrained access to the entire brain, impeding the exploration of brain networks crucial at various stages of this paradigm. Recently, advances in functional magnetic resonance imaging (fMRI) in the avian brain have opened new windows to explore bird's brain function at the network level. Here, we developed a ground-breaking non-invasive functional MRI technique for awake, newly hatched chicks that record whole-brain BOLD signal changes throughout imprinting experiments. While the initial phases of memory acquisition imprinting behaviour have been unravelled, the long-term storage and retrieval components of imprinting memories are still unknown. Our findings identified potential long-term storage of imprinting memories across a neural network, including the hippocampal formation, the medial striatum, the arcopallium, and the prefrontal-like nidopallium caudolaterale. This platform opens up new avenues for exploring the broader landscape of learning and memory processes in neonatal vertebrates, contributing to a more comprehensive understanding of the intricate interplay between behaviour and brain networks.

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.

The domestic chick as an animal model of autism spectrum disorder: building adaptive social perceptions through prenatally formed predispositions.

Equipped with an early social predisposition immediately post-birth, humans typically form associations with mothers and other family members through exposure learning, canalized by a prenatally formed predisposition of visual preference to biological motion, face configuration, and other cues of animacy. If impaired, reduced preferences can lead to social interaction impairments such as autism spectrum disorder (ASD) via misguided canalization. Despite being taxonomically distant, domestic chicks could also follow a homologous developmental trajectory toward adaptive socialization through imprinting, which is guided via predisposed preferences similar to those of humans, thereby suggesting that chicks are a valid animal model of ASD. In addition to the phenotypic similarities in predisposition with human newborns, accumulating evidence on the responsible molecular mechanisms suggests the construct validity of the chick model. Considering the recent progress in the evo-devo studies in vertebrates, we reviewed the advantages and limitations of the chick model of developmental mental diseases in humans.

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.

Innate sensitivity to face-to-face biological motion.

Sensitivity to face-to-face stimuli configurations, which likely indicates interaction, seems to appear early in infants' development, and recently a preference for face-to-face (vs. other spatial configurations) has been shown to occur in macaque monkeys. It is unknown, however, whether such a preference is acquired through experience or as an evolutionary-given biological predisposition. Here, we exploited a precocial social animal, the domestic chick, as a model system to address this question. Visually naive chicks were tested for their spontaneous preferences for face-to-face vs. back-to-back hen dyads of point-light displays depicting biological motion. We found that female chicks have a spontaneous preference for the facing interactive configuration. Males showed no preference, as expected due to the well-known low social motivation of males in this highly polygynous species. These findings support the idea of an innate and sex-dependent predisposition toward social and interacting stimuli in a vertebrate brain such as that of chicks.

Transfer from continuous to discrete quantities in honeybees.

Honeybees can estimate quantities having different dimensions: continuous and uncountable such as the relative size of visual objects in an array, or discrete and countable such as the number of objects of the array. Honeybees can transfer quantity discrimination (i.e., choosing the larger/smaller stimulus) from number to size. Here, we investigated whether honeybees could also generalize from the size (continuous) to the number (discrete) dimension. We trained free-flying foragers to discriminate between large- and small-size elements. At test, bees were presented with a comparison between larger and smaller numerosities controlled for different continuous variables covarying with numerosity such as total area, total perimeter, convex hull, and element size. Results showed that bees generalized from the size to the numerical dimension of the stimuli. This cross-dimensional transfer supports the idea of a universal mechanism for the encoding of abstract magnitudes in invertebrate species comparable to that of vertebrate species.

Manganese-enhanced magnetic resonance imaging reveals light-induced brain asymmetry in embryo.

The idea that sensory stimulation to the embryo (in utero or in ovo) may be crucial for brain development is widespread. Unfortunately, up to now evidence was only indirect because mapping of embryonic brain activity in vivo is challenging. Here, we applied for the first time manganese enhanced magnetic resonance imaging (MEMRI), a functional imaging method, to the eggs of domestic chicks. We revealed light-induced brain asymmetry by comparing embryonic brain activity in vivo of eggs that were stimulated by light or maintained in the darkness. Our protocol paves the way to investigation of the effects of a variety of sensory stimulations on brain activity in embryo.

Neural coding of numerousness.

Perception of numerousness, i.e. number of items in a set, is an important cognitive ability, which is present in several animal taxa. In spite of obvious differences in neuroanatomy, insects, fishes, reptiles, birds, and mammals all possess a "number sense". Furthermore, information regarding numbers can belong to different sensory modalities: animals can estimate a number of visual items, a number of tones, or a number of their own movements. Given both the heterogeneity of stimuli and of the brains processing these stimuli, it is hard to imagine that number cognition can be traced back to the same evolutionary conserved neural pathway. However, neurons that selectively respond to the number of stimuli have been described in higher-order integration brain centres both in primates and in birds, two evolutionary distant groups. Although most probably not of the same evolutionary origin, these number neurons share remarkable similarities in their response properties. Instead of homology, this similarity might result from computational advantages of the underlying coding mechanism. This means that one might expect numerousness information to undergo similar steps of neural processing even in evolutionary distant neural pathways. Following this logic, in this review we summarize our current knowledge of how numerousness is processed in the brain from sensory input to coding of abstract information in the higher-order integration centres. We also propose a list of key open questions that might promote future research on number cognition.

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".

Life is in motion (through a chick's eye).

Cognitive scientists, social psychologists, computer scientists, neuroscientists, ethologists and many others have all wondered how brains detect and interpret the motion of living organisms. It appears that specific cues, incorporated into our brains by natural selection, serve to signal the presence of living organisms. A simple geometric figure such as a triangle put in motion with specific kinematic rules can look alive, and it can even seem to have intentions and goals. In this article, we survey decades of parallel investigations on the motion cues that drive animacy perception-the sensation that something is alive-in non-human animals, especially in precocial species, such as the domestic chick, to identify inborn biological predispositions. At the same time, we highlight the relevance of these studies for an understanding of human typical and atypical cognitive development.

baz1b loss-of-function in zebrafish produces phenotypic alterations consistent with the domestication syndrome.

BAZ1B is a ubiquitously expressed nuclear protein with roles in chromatin remodeling, DNA replication and repair, and transcription. Reduced BAZ1B expression disrupts neuronal and neural crest development. Variation in the activity of BAZ1B has been proposed to underly morphological and behavioral aspects of domestication through disruption of neural crest development. Knockdown of baz1b in Xenopus embryos and Baz1b loss-of-function (LoF) in mice leads to craniofacial defects consistent with this hypothesis. We generated baz1b LoF zebrafish using CRISPR/Cas9 gene editing to test the hypothesis that baz1b regulates behavioral phenotypes associated with domestication in addition to craniofacial features. Zebrafish with baz1b LoF show mild underdevelopment at larval stages and distinctive craniofacial features later in life. Mutant zebrafish show reduced anxiety-associated phenotypes and an altered ontogeny of social behaviors. Thus, in zebrafish, developmental deficits in baz1b recapitulate both morphological and behavioral phenotypes associated with the domestication syndrome in other species.

Efficient Low-Frequency SSVEP Detection with Wearable EEG Using Normalized Canonical Correlation Analysis.

Recent studies show that the integrity of core perceptual and cognitive functions may be tested in a short time with Steady-State Visual Evoked Potentials (SSVEP) with low stimulation frequencies, between 1 and 10 Hz. Wearable EEG systems provide unique opportunities to test these brain functions on diverse populations in out-of-the-lab conditions. However, they also pose significant challenges as the number of EEG channels is typically limited, and the recording conditions might induce high noise levels, particularly for low frequencies. Here we tested the performance of Normalized Canonical Correlation Analysis (NCCA), a frequency-normalized version of CCA, to quantify SSVEP from wearable EEG data with stimulation frequencies ranging from 1 to 10 Hz. We validated NCCA on data collected with an 8-channel wearable wireless EEG system based on BioWolf, a compact, ultra-light, ultra-low-power recording platform. The results show that NCCA correctly and rapidly detects SSVEP at the stimulation frequency within a few cycles of stimulation, even at the lowest frequency (4 s recordings are sufficient for a stimulation frequency of 1 Hz), outperforming a state-of-the-art normalized power spectral measure. Importantly, no preliminary artifact correction or channel selection was required. Potential applications of these results to research and clinical studies are discussed.

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.

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.

Number neurons in the nidopallium of young domestic chicks.

Numerical cognition is ubiquitous in the animal kingdom. Domestic chicks are a widely used developmental model for studying numerical cognition. Soon after hatching, chicks can perform sophisticated numerical tasks. Nevertheless, the neural basis of their numerical abilities has remained unknown. Here, we describe number neurons in the caudal nidopallium (functionally equivalent to the mammalian prefrontal cortex) of young domestic chicks. Number neurons that we found in young chicks showed remarkable similarities to those in the prefrontal cortex and caudal nidopallium of adult animals. Thus, our results suggest that numerosity perception based on number neurons might be an inborn feature of the vertebrate brain.

Light-induced asymmetries in embryonic retinal gene expression are mediated by the vascular system and extracellular matrix.

Left-right asymmetries in the nervous system (lateralisation) influence a broad range of behaviours, from social responses to navigation and language. The role and pathways of endogenous and environmental mechanisms in the ontogeny of lateralisation remains to be established. The domestic chick is a model of both endogenous and experience-induced lateralisation driven by light exposure. Following the endogenous rightward rotation of the embryo, the asymmetrical position in the egg results in a greater exposure of the right eye to environmental light. To identify the genetic pathways activated by asymmetric light stimulation, and their time course, we exposed embryos to different light regimes: darkness, 6 h of light and 24 h of light. We used RNA-seq to compare gene expression in the right and left retinas and telencephalon. We detected differential gene expression in right vs left retina after 6 h of light exposure. This difference was absent in the darkness condition and had already disappeared by 24 h of light exposure, suggesting that light-induced activation is a self-terminating phenomenon. This transient effect of light exposure was associated with a downregulation of the sensitive-period mediator gene DIO2 (iodothyronine deiodinase 2) in the right retina. No differences between genes expressed in the right vs. left telencephalon were detected. Gene networks associated with lateralisation were connected to vascularisation, cell motility, and the extracellular matrix. Interestingly, we know that the extracellular matrix-including the differentially expressed PDGFRB gene-is involved in morphogenesis, sensitive periods, and in the endogenous chiral mechanism of primary cilia, that drives lateralisation. Our data show a similarity between endogenous and experience-driven lateralisation, identifying functional gene networks that affect lateralisation in a specific time window.

Young domestic chicks spontaneously represent the absence of objects.

Absence is a notion that is usually captured by language-related concepts like zero or negation. Whether nonlinguistic creatures encode similar thoughts is an open question, as everyday behavior marked by absence (of food, of social partners) can be explained solely by expecting presence somewhere else. We investigated 8-day-old chicks' looking behavior in response to events violating expectations about the presence or absence of an object. We found different behavioral responses to violations of presence and absence, suggesting distinct underlying mechanisms. Importantly, chicks displayed an avian signature of novelty detection to violations of absence, namely a sex-dependent left-eye-bias. Follow-up experiments excluded accounts that would explain this bias by perceptual mismatch or by representing the object at different locations. These results suggest that the ability to spontaneously form representations about the absence of objects likely belongs to the initial cognitive repertoire of vertebrate species.

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.

Archerfish number discrimination.

Debates have arisen as to whether non-human animals actually can learn abstract non-symbolic numerousness or whether they always rely on some continuous physical aspect of the stimuli, covarying with number. Here, we investigated archerfish (Toxotes jaculatrix) non-symbolic numerical discrimination with accurate control for covarying continuous physical stimulus attributes. Archerfish were trained to select one of two groups of black dots (Exp. 1: 3 vs 6 elements; Exp. 2: 2 vs 3 elements); these were controlled for several combinations of physical variables (elements' size, overall area, overall perimeter, density, and sparsity), ensuring that only numerical information was available. Generalization tests with novel numerical comparisons (2 vs 3, 5 vs 8, and 6 vs 9 in Exp. 1; 3 vs 4, 3 vs 6 in Exp. 2) revealed choice for the largest or smallest numerical group according to the relative number that was rewarded at training. None of the continuous physical variables, including spatial frequency, were affecting archerfish performance. Results provide evidence that archerfish spontaneously use abstract relative numerical information for both small and large numbers when only numerical cues are available.