Biological predispositions to orient to and preferentially learn about conspecifics are one of the earliest expressions of social behavior in vertebrates and are critical for survival. These elementary behavioral markers of social orienting are spontaneous, possibly hard-wired, mechanisms that bias visual attention to simple features of animate beings since the earliest minutes of life (Goren et al., 1975; Johnson et al., 1991). Human faces and schematic face-like patterns generate remarkable responses in typical developing neonates (Simion and Di Giorgio, 2015). More strikingly, the same abilities can be observed in newly-hatched chicks (Rosa-Salva et al., 2010; Rosa Salva et al., 2011) and visually naïve monkeys (Sugita, 2008, 2009). Other species have also been shown to respond to similar schematic configurations (Leopold and Rhodes, 2010), such that privileged face processing could be pervasive in vertebrates.

More importantly, it has been hypothesized that early disturbances of these social orienting mechanisms may be one of the earliest signs of social deficits in autism spectrum disorders (ASD) and might also contribute to the pathophysiology of these disorders by compromising, early on, the typical developmental trajectories of the social brain (Dawson et al., 2005; Johnson, 2005; Senju and Johnson, 2009; Johnson et al., 2015). In line with that, impairments in face and eye-gaze direction processing have been reported in infants at risk of ASD (Di Giorgio et al., 2016; Webb et al., 2017, for a critical discussion see also Jones and Klin, 2013; Shultz et al., 2018; Bradshaw et al., 2020).

Given the complexity of human social behavior and the limitations that human studies impose, animal models are instrumental in providing clues on the nature and origin of these crucial social orienting mechanisms and their role in atypical social development. Valproic acid (VPA) exposure has been extensively used in several animal models to reproduce ASD core symptoms (Bambini-Junior et al., 2014). Previous studies have shown that exposure to different doses of VPA during embryogenesis induces alterations of several aspects of social behavior in domestic chicks (Nishigori et al., 2013; Zachar et al., 2019). We used VPA exposure to induce neurodevelopmental changes associated with social deficits in domestic chicks and tested whether VPA could impact the expression of early approach responses to schematic face-like patterns. We found that VPA impairs the chicks’ preference responses to these social stimuli. Based on the results shown here, we propose the domestic chicks as elective animal models to study these early-emerging neurobehavioral markers and to investigate the biological mechanisms underlying face processing deficits in ASD.

To assess the effect of VPA on face perception, and avoid any possible influence of previous experiences in evaluating the chicks’ approach to the stimuli, we excluded visual experience prior to the test. To obtain a better approach rate, we extended the duration of the test compared to the previous reports to 12 mins. Using this adapted paradigm, we tested 69 chicks (31 females, 38 males), 24 h after hatching.

We first analyzed the time spent by the animals in the choice sections of the apparatus (Figure 2A) using a mixed model analysis (see “Materials and Methods”). The results showed no significant main effect of treatment and sex on the time spent in the apparatus sections [treatment F_{(1, 195)} = 4.812E-012, p > 0.9999; sex F_{(1, 195)} = 1.084E-010, p > 0.9999], a significant main effect of the sections [apparatus sections F_{(2, 195)} = 44.48, p < 0.0001] and a significant interaction of the treatment on the visited sections [treatment × apparatus sections F_{(2, 195)} = 4.904, p = 0.0084]. No other significant interactions emerged [treatment × sex F_{(1, 195)} = 9.114E-011, p > 0.9999; sex × apparatus sections F_{(2, 195)} = 0.4469, p = 0.6403; treatment × sex × apparatus sections F_{(2, 195)} = 1.287, p = 0.2784]. The Sidak multiple comparison test showed a significant effect of treatment on the time spent in the non-face chamber [t₍₂₀₁₎ = 2.421, p = 0.0335]. Thus, VPA treatment selectively increases the time spent by the animals attending the non-face stimulus.

To further evaluate the effect of treatment on the preference for the stimuli independent of the exploratory activity we also analyzed the effect of VPA exposure on the preference index (see “Materials and Methods”). We found a significant difference between the treatment groups in the preference index for the schematic face-like configuration stimulus [Figure 2B; treatment: F_{(1, 65)} = 4.805, p = 0.0320; sex: F_{(1, 65)} = 0.5745, p = 0.4512; treatment × sex: F_{(1, 65)} = 2.652, p = 0.1083]. While vehicle-injected chicks significantly preferred the schematic face-like stimulus, VPA-exposed chicks did not display any significant preference for this stimulus compared to what expected by chance [Figure 2B; CTRL t₍₃₂₎ = 2.481, p = 0.0186; VPA t₍₃₅₎ = 0.3425, p = 0.7341; group mean: CTRL 0.6694 (95% CI: 0.5303–0.8085); VPA 0.4764 (95% CI: 0.3364–0.6164)]. We then analyzed the latency to express a choice and the number of section alternations after the first choice. We found a significant effect of treatment on the latency: VPA-injected chicks had a shorter latency to choice compared to controls [Figure 2C; treatment: F_{(1, 65)} = 5.369, p = 0.0237 sex: F_{(1, 65)} = 0.1881, p = 0.6660; treatment × sex: F_{(1, 65)} = 0.1270, p = 0.7228; group mean: CTRL 339 s (95% CI: 275–403); VPA 234 s (95% CI: 172–295)]. Spontaneous alternations in the two choice sections did not significantly differ between treatment groups [Figure 2D; treatment: F_{(1, 65)} = 1.941, p = 0.1683; sex: F_{(1, 65)} = 0.0790, p = 0.7795; treatment × sex: F_{(1, 65)} = 1.293, p = 0.2598; group mean: CTRL 5.091 (95% CI: 2.344–7.838); VPA 8.389 (95% CI: 5.077–11.70)].

The number of chicks that approached the face-like configuration as the first stimulus was not significantly different between treatment groups (Pearson’s X₁² = 2.944, p = 0.0862; CTRL: face N = 21, non-face N = 12, VPA: face N = 15, non-face N = 20, data not shown).

Newborns of several vertebrate species exhibit rudimental knowledge about the typical appearance of animate beings that orients the young organisms’ attention toward plausible social partners and caregivers. Several studies hypothesized that this mechanism contributes to create an early social bond with caretakers and social companions (Johnson, 2005; Tomalski et al., 2009), an essential process for subsequent social and language development. Newborn babies, as well as non-human primates and domestic chicks, have been shown to express remarkable orienting responses to faces and schematic face-like patterns (Sugita, 2008, 2009; Rosa-Salva et al., 2010; Rosa Salva et al., 2011). Divergence from these early social interactions may induce a cascade of maladaptive trajectories culminating in atypical social abilities, such as those observed in ASD.

Predispositions in domestic chicks have been observed toward a variety of features of animate creatures and trigger preference responses to a very broad spectrum of representations: being them face-like configurations (Rosa Salva et al., 2011) biological motion (Vallortigara et al., 2005) or self-propelled motion (Rosa-Salva et al., 2016). Newly hatched domestic chicks express social preferences to features of animals belonging to other species, including potential predators, as shown by their innate preference toward a walking cat (Vallortigara et al., 2005) represented by point light displays or toward a taxidermized polecat (Rosa-Salva et al., 2019) or a human face (Rosa Salva et al., 2011). Similarly, face-naïve Japanese macaques spend equal time attending to humans and monkey faces and prefer both over inanimate objects (Sugita, 2008). This data shows that biological predispositions are clearly not species-specific, but include rudimental configurations shared across species to increase the chance of orienting toward other animals. In the natural environment of a newly hatched organism, these other animals are most likely to be conspecifics (parents, siblings). Subsequently activated learning mechanisms, whose action is directed toward living creatures by the predispositions themselves, will provide the young animals with species-specific information on the appearance of their conspecifics [see also Morton and Johnson (1991) and Johnson (2005) for a broader discussion of the species-general nature of the representations underlying face-preferences in newborn babies and domestic chicks]. As to whether the face-like stimulus can be extended as a feature of conspecifics, studies show that the predisposed preference observed in newly-hatched chicks toward the stuffed hen or taxidermized newly-hatched chicks, mallard ducks or polecats, are indeed triggered by the head and neck region, suggesting a major role of face configurations in the head region (Johnson and Horn, 1988; Rosa-Salva et al., 2019; Miura et al., 2020).

Using the preference response to face-like stimuli as an evolutionarily conserved neurobehavioral marker and exploiting the advantages of animal models, we investigated whether these early-emerging social orienting mechanisms could be affected by a compound, VPA, known to interfere with development of the social brain. We examined the preference response toward schematic-face like configurations of animals whose pattern of brain development may have been altered by VPA, an anticonvulsant increasing the risk to develop ASD in humans. We found that VPA had a dramatic effect on the preference toward schematic-face configuration stimuli.

Previous studies have revealed a predisposed response to schematic face-like configurations in newly-hatched chicks, using both subjects imprinted on face-neutral stimuli and visually naïve subjects (Rosa-Salva et al., 2010; Rosa Salva et al., 2011, 2012). To assess the effect of VPA on face perception, and avoid any possible influence of previous experiences on the chicks’ approach to the stimuli, we applied this latter experimental procedure, excluding visual experience prior to the test. Since dark reared animals are less active compared to chicks exposed to visual stimuli, to obtain a better approach rate, we extended the duration of the test compared to the previous reports. Increasing the test duration in our experiment contributed to heighten the approach response and the face preference, without introducing the potential influence of visual experience. We also noticed that the preference for the face-like stimulus was especially conspicuous in control females, which showed a remarkable preference level compared to all other groups [Figure 3; group mean preference index CTRL females 0.8029 (95% CI: 0.6425–0.9632), one-sample t-test t₍₁₃₎ = 4.081, uncorrected p = 0.0013; group mean preference index VPA females 0.4318 (95% CI: 0.2064–0.6571); t₍₁₂₎ = 0.6419, uncorrected p = 0.5301; group mean preference index CTRL males 0.5711 (95% CI: 0.3589–0.7832); t₍₁₈₎ = 0.7035, uncorrected p = 0.4907; group mean preference index VPA males 0.5163 (95% CI: 0.3245–0.7081); t₍₁₈₎ = 0.1787, uncorrected p = 0.8602]. However, given that no significant interaction between the factors sex and treatment emerged in our previous analysis, any difference between the two sexes observed here should be interpreted with caution. Notably, regardless of the sex of the chicks examined, VPA-exposed chicks spent significantly more time attending the non-social stimulus. This data is in line with what observed in other VPA models (Zhao et al., 2019) in which juvenile VPA-treated monkeys attended to non-social stimuli significantly more than their control siblings. Future studies will investigate the potential sex differences in the level of face-preference and in their susceptibility to VPA, suggested by some of our data, and clarify the mechanism of action of VPA on the development and expression of face preference in domestic chicks.

The reduced latencies observed in the VPA group, indicate that VPA exposure affects the visual preference for schematic face-configuration patterns without significantly hindering the chicks’ motoric activity during the test. In line with that, previous studies from our lab have shown that VPA exposure, at the dosage used in this study, does not significantly affect motor behavior or discriminative abilities of simple artificial objects in domestic chicks (Sgadò et al., 2018).

A previous study has investigated the attentive behavior toward faces in VPA-exposed juvenile macaques (Zhao et al., 2019). Using eye-tracking analysis to measure the animals’ attention to faces or scene containing conspecifics, the authors found that juvenile VPA-treated monkeys attended to non-social stimuli significantly more than their control siblings. However, the study did not specifically investigate the predisposed response of visually naïve animals to faces compared to a visually equivalent stimulus without social content. In this respect, our study is the first to analyze a very early predisposed response to faces in a visually naïve animal model of ASD.

Valproic acid is an anticonvulsant extensively used to treat epilepsy and bipolar disorders. VPA mechanism of action involves its direct inhibition of histone deacetylases (HDACs), interfering with normal deacetylation of chromatin and disrupting gene transcription at global scale, as well as HDAC independent mechanisms (Sinha et al., 2021). Embryonic exposure to VPA is normally achieved by a single acute dose of VPA (ranging between 400 and 800 mg/kg in rodents) that induces a transient HDAC inhibition producing long lasting effects. Several studies suggest that embryonic VPA exposure affects neurogenesis (Kataoka et al., 2013; Lee et al., 2016; Sakai et al., 2018; Zhao et al., 2019; Cui et al., 2020; Sawada et al., 2021) and alters expression of several neurodevelopmental genes, involving serotonergic system development (Jacob et al., 2014; Messina et al., 2020) and excitation/inhibition imbalance (Rinaldi et al., 2007; Gogolla et al., 2009; Banerjee et al., 2013; Nagode et al., 2017). Given its antiepileptic pharmacological action, VPA has been shown to increase GABA levels in the brain, trough different mechanisms, acting on GABA transaminase and other enzymes linked to the metabolism of GABA (Johannessen, 2000), as well as trough inhibition of sodium channels (Abdelsayed and Sokolov, 2013). Despite extensive research investigating VPA pharmacological action and the genetic networks responsible for its effects on brain development, the biological mechanisms underlying the detrimental consequences of embryonic VPA exposure on social behavior in animal models are still unclear.

Altogether, this study and previous studies from our lab, demonstrate a detrimental effect of VPA, an anticonvulsant increasing the risk to develop ASD in humans, on the very early predisposed responses toward social stimuli in visually-naïve domestic chicks. Based on these results, we propose the domestic chicks as elective animal models to study these early-emerging neurobehavioral markers and to investigate the biological mechanisms underlying face processing deficits in ASD.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Ethical Committee of the University of Trento and licensed by the Italian Health Ministry (permit number 986/2016-PR).

PS conceived and designed the experiments and drafted the manuscript. AA and SP conducted the experiments. PS and OR-S analyzed the data. AA, PS, and OR-S wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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