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.

Temporal hampering of thyroid hormone synthesis just before hatching impeded the filial imprinting in domestic chicks.

Thyroid hormones play a critical role in the initiation of the sensitive period of filial imprinting. The amount of thyroid hormones in the brains of chicks increases intrinsically during the late embryonic stages and peaks immediately before hatching. After hatching, a rapid imprinting-dependent inflow of circulating thyroid hormones into the brain occurs via vascular endothelial cells during imprinting training. In our previous study, inhibition of hormonal inflow impeded imprinting, indicating that the learning-dependent inflow of thyroid hormones after hatching is critical for the acquisition of imprinting. However, it remained unclear whether the intrinsic thyroid hormone level just before hatching affects imprinting. Here, we examined the effect of temporal thyroid hormone decrease on embryonic day 20 on approach behavior during imprinting training and preference for the imprinting object. To this end, methimazole (MMI; a thyroid hormone biosynthesis inhibitor) was administered to the embryos once a day on days 18-20. Serum thyroxine (T4) was measured to evaluate the effect of MMI. In the MMI-administered embryos, the T4 concentration was transiently reduced on embryonic day 20 but recovered to the control level on post-hatch day 0. At the beginning of imprinting training on post-hatch day 1, control chicks approached the imprinting object only when the object was moving. In the late phase of training, control chicks subsequently approached towards the static imprinting object. On the other hand, in the MMI-administered chicks, the approach behavior decreased during the repeated trials in the training, and the behavioral responses to the imprinting object were significantly lower than those of control chicks. This indicates that their persistent responses to the imprinting object were impeded by a temporal thyroid hormone decrease just before hatching. Consequently, the preference scores of MMI-administered chicks were significantly lower than those of control chicks. Furthermore, the preference score on the test was significantly correlated with the behavioral responses to the static imprinting object in the training. These results indicate that the intrinsic thyroid hormone level immediately before hatching is crucial for the learning process of imprinting.

Suppressive Modulation of the Chick Forebrain Network for Imprinting by Thyroid Hormone: An in Vitro Study.

The thyroid hormone 3,5,3'-triiodothyronine (T3) is considered to act acutely in the chick forebrain because focal infusion of T3 to the intermediate medial mesopallium (IMM) causes 4 to 6-day-old hatchlings to become imprintable approximately 30 min after the infusion. To understand the mechanism of this acute T3 action, we examined synaptic responses of IMM neurons in slice preparations in vitro. Extracellular field potential responses to local electrical stimulation were pharmacologically dissociated to synaptic components mediated by AMPA and NMDA receptors, as well as GABA-A and -B receptors. Bath-applied T3 (20-40 µM) enhanced the positive peak amplitude of the field potential, which represented the GABA-A component. Bicuculline induced spontaneous epileptic bursts by NMDA receptor activation, and subsequent application of T3 suppressed the bursting frequency. Pretreatment of slices with T3 failed to influence the synaptic potentiation caused by tetanic stimulation. Intracellular whole-cell recording using a patch electrode confirmed the T3 actions on the GABA-A and NMDA components. T3 enhanced the GABA-A response and suppressed the NMDA plateau potential without changes in the resting membrane potential or the threshold of action potentials. Contrary to our initial expectation, T3 suppressed the synaptic drives of IMM neurons, and did not influence activity-dependent synaptic potentiation. Imprinting-associated T3 influx may act as an acute suppressor of the IMM network.

Chick Hippocampal Formation Displays Subdivision- and Layer-Selective Expression Patterns of Serotonin Receptor Subfamily Genes.

Hippocampal formation (HF) plays a key role in cognitive and emotional processing in mammals. In HF neural circuits, serotonin receptors (5-HTRs) modulate functions related to cognition and emotion. To understand the phylogenetic continuity of the neural basis for cognition and emotion, it is important to identify the neural circuits that regulate cognitive and emotional processing in animals. In birds, HF has been shown to be related to cognitive functions and emotion-related behaviors. However, details regarding the distribution of 5-HTRs in the avian brain are very sparse, and 5-HTRs, which are potentially involved in cognitive functions and emotion-related behaviors, are poorly understood. Previously, we showed that 5-HTR1B and 5-HTR3A were expressed in chick HF. To identify additional 5-HTRs that are potentially involved in cognitive and emotional functions in avian HF, we selected the chick orthologs of 5-HTR1D, 5-HTR1E, 5-HTR1F, 5-HTR2B, 5-HTR5A, and 5-HTR7 and performed in situ hybridization in the chick telencephalon. We found that 5-HTR1D, 5-HTR1E, 5-HTR5A, and 5-HTR7 were expressed in the chick HF, especially 5-HTR1D and 5-HTR1E, which showed subdivision- and layer-selective expression patterns, suggesting that the characteristic 5-HT regulation is involved in cognitive functions and emotion-related behaviors in these HF regions. These findings can facilitate the understanding of serotonin regulation in avian HF and the correspondence between the HF subdivisions of birds and mammals.

Imprintability of Newly Hatched Domestic Chicks on an Artificial Object: A Novel High Time-Resolution Apparatus Based on a Running Disc.

In filial imprinting, newly hatched chicks repeatedly approach a conspicuous object nearby and memorize it, even though it is an artificial object instead of their mother hen. Imprinting on an artificial object in a laboratory setting has a clear sensitive period from post hatch days 1-3 in the case of domestic chicks. However, the establishment of imprintability are difficult to investigate because of the limitations of the behavioral apparatus. In this study, we developed a novel behavioral apparatus, based on a running disc, to investigate the learning processes of imprinting in newly hatched domestic chicks. In the apparatus, the chick repeatedly approaches the imprinting object on the disc. The apparatus sends a transistor-transistor-logic signal every 1/10 turn of the disc to a personal computer through a data acquisition system following the chick's approach to the imprinting object on the monitor. The imprinting training and tests were designed to define the three learning processes in imprinting. The first process is the one in which chicks spontaneously approach the moving object. The second is an acquired process in which chicks approach an object even when it is static. In the third process, chicks discriminate between the differently colored imprinting object and the control object in the preference test. Using the apparatus, the difference in the chicks' behavior during or after the sensitive period was examined. During the sensitive period, the chicks at post hatch hour 12 and 18 developed the first imprinting training process. The chicks at post hatch hour 24 maintained learning until the second process. The chicks at post hatch hour 30 reached the discrimination process in the test. After the sensitive period, the chicks reared in darkness until post hatch day 4 exhibited poor first learning process in the training. Thus, this apparatus will be useful for the detection of behavioral changes during neuronal development and learning processes.

Subtype-selective contribution of muscarinic acetylcholine receptors for filial imprinting in newly-hatched domestic chicks.

Muscarinic acetylcholine receptors (mAChRs) play an important role in many brain functions. Our previous study revealed that the injection of mAChRs antagonist scopolamine into the intermediate medial mesopallium (IMM) region, which is critical for filial imprinting, impairs memory formation. In avian brains, four mAChR subtypes have been identified (M2, M3, M4 and M5). M3 and M5 receptors increase the excitability of neurons, whereas M2 and M4 receptors reduce the excitability. Because the scopolamine blocks all subtypes, the previous study did not identify which subtype contributes to the memory formation. By injecting several types of mAChR antagonists into the IMM, in this study we determined which mAChR subtype plays a critical role in imprinting. First, the effects of antagonists on the excitatory receptor subtypes M3 and M5 were examined. Injection of the M3 antagonist (DAU5884) at 20 mM or the M5 antagonist (ML381) at 2 mM impaired imprinting. Considering the pKi value of DAU5884, the impairment seems to be caused by DAU5884 binding to M3 and/or M4 receptors. Second, the effect of antagonists on the inhibitory receptor subtype M2 was examined. The results showed that the M2 antagonist (AQ-RA741) impaired imprinting at a concentration of 20 mM. Considering the pKi value of AQ-RA741, the impairment seems to be caused by AQ-RA741 binding to M2 and/or M4. The findings of this study suggests that the excitatory receptor subtypes M3 and M5 and the inhibitory receptor subtype M2 and/or M4 cooperate to achieve the appropriate balance of acetylcholine signaling to execute imprinting.

Fetal blockade of nicotinic acetylcholine transmission causes autism-like impairment of biological motion preference in the neonatal chick.

Several environmental chemicals are suspected risk factors for autism spectrum disorder (ASD), including valproic acid (VPA) and pesticides acting on nicotinic acetylcholine receptors (nAChRs), if administered during pregnancy. However, their target processes in fetal neuro-development are unknown. We report that the injection of VPA into the fetus impaired imprinting to an artificial object in neonatal chicks, while a predisposed preference for biological motion (BM) remained intact. Blockade of nAChRs acted oppositely, sparing imprinting and impairing BM preference. Beside ketamine and tubocurarine, significant effects of imidacloprid (a neonicotinoid insecticide) appeared at a dose ≤1 ppm. In accord with the behavioral dissociations, VPA enhanced histone acetylation in the primary cell culture of fetal telencephalon, whereas ketamine did not. VPA reduced the brain weight and the ratio of NeuN-positive cells (matured neurons) in the telencephalon of hatchlings, whereas ketamine/tubocurarine did not. Despite the distinct underlying mechanisms, both VPA and nAChR blockade similarly impaired imprinting to biological image composed of point-light animations. Furthermore, both impairments were abolished by postnatal bumetanide treatment, suggesting a common pathology underlying the social attachment malformation. Neurotransmission via nAChR is thus critical for the early social bond formation, which is hindered by ambient neonicotinoids through impaired visual predispositions for animate objects.

Gene expression profiles of the muscarinic acetylcholine receptors in brain regions relating to filial imprinting of newly-hatched domestic chicks.

Muscarinic acetylcholine receptors (mAChRs) in the central nervous system play an important role in regulating complex functions such as learning, memory, and selective attention. Five subtypes of the mAChRs (M1-M5) have been identified in mammals, and are classified into two subfamilies: excitatory (M1, M3, and M5) and inhibitory (M2 and M4) subfamilies. Filial imprinting of domestic chicks is a useful model in the laboratory to investigate the mechanisms of memory formation in early learning. We recently found that mAChRs in the intermediate medial mesopallium (IMM) are involved in the memory formation of imprinting. However, expression profiles of each mAChR subtype in the brain regions including the IMM remain unexplored. Here we show the unique gene expression of each mAChR subtype in the pallial regions involved in imprinting. In terms of the excitatory mAChRs, M5 was expressed in the IMM region and other parts of the pallium, whereas M3 was less expressed in the IMM but highly expressed in the hyperpallium and nidopallium. Regarding the inhibitory mAChRs, M2 was sparsely distributed but clearly in some cells throughout the pallial regions. M4 was highly expressed in the IMM region and other parts of the pallium. These expression profiles can be used as a basis for understanding cholinergic modulation in the memory formation of imprinting and other learning processes in birds, and compared to those of mammals.

Construction of Roman roads toward neuroeconomics.

Neuroeconomics is still "under construction." To be a leading discipline, it needs firm ecological rationale and neurobiological bases. "Vigor" supplies this infrastructure through the mathematics of the foraging theory and system-neuroscience evidence on utility and motor control. It will prepare us for the future neuroeconomics, if studied appropriately in the light of evolution.

Resurgence of an Inborn Attraction for Animate Objects via Thyroid Hormone T3.

For inexperienced brains, some stimuli are more attractive than others. Human neonates and newly hatched chicks preferentially orient towards face-like stimuli, biological motion, and objects changing speed. In chicks, this enhances exposure to social partners, and subsequent attachment trough filial imprinting. Early preferences are not steady. For instance, preference for stimuli changing speed fades away after 2 days in chicks. To understand the physiological mechanisms underlying these transient responses, we tested whether early preferences for objects changing speed can be promoted by thyroid hormone 3,5,3'-triiodothyronine (T3). This hormone determines the start of imprinting's sensitive period. We found that the preference for objects changing speed can be re-established in female chicks treated with T3. Moreover, day-1 chicks treated with an inhibitor of endogenous T3 did not show any preference. These results suggest that the time windows of early predispositions and of sensitive period for imprinting are controlled by the same molecular mechanisms.

What Do Adolescents Know About One-Health and Zoonotic Risks? A School-Based Survey in Italy, Austria, Germany, Slovenia, Mauritius, and Japan.

More than 60% of the 1,700 infectious diseases that affect human come from animals and zoonotic pandemics, after starting from sporadic phenomena limited to rural areas, have become a global emergency. The repeated and frequent zoonotic outbreaks such as the most recent COVID-19 pandemic can be attributed also to human activities. In particular, the creation of enormous intensive domestic animal farms, the indiscriminate use of antibiotics, the destruction of forests, the consumption of the meat of wild animals and the illegal animal trade are all factors causing the insurgence and the transmission of zoonotic diseases from animals to humans. The purpose of this study was to explore the knowledge of the One Health concept including the zoonotic risk potentially derived from illegally traded pet animals and wildlife among adolescents in 6 different countries (Italy, Austria, Slovenia, Germany, Mauritius, and Japan). A representative sample of 656 students was recruited and all participants took an anonymous questionnaire. Data were analyzed by ANOVAs to estimate the prevalence of correct health prevention behaviors and to identify the influential factors for these behaviors. After two theoretical-practical lectures, the same anonymous questionnaire was administered for the second time in order to assess the efficacy of the program. The proportion of students who did not know that many diseases affecting humans come from animals is 28.96% while 32.16% of them did not know what a zoonosis is. The circularity of the One Health concept related to the transmission of diseases from animals to humans and vice-versa is not understood from a large prevalence of the adolescents with 31.40 and 59.91% of wrong responses, respectively. Furthermore, rabies is not considered as a dangerous disease by 23.02% of the adolescents. After two theoretical-practical classroom sessions, the correct answers improved to 21.92% according to the different question. More than a third of the student cohort investigated showed a profound ignorance of the zoonotic risks and a poor understanding of the One Health concept. The authors believe that the teaching of health prevention with a One Health approach and a practical training should be included in every school curriculum.

Serotonergic Neurons in the Chick Brainstem Express Various Serotonin Receptor Subfamily Genes.

Serotonin (5-hydroxytryptamine, 5-HT) is a phylogenetically conserved modulatory neurotransmitter. In mammals, 5-HT plays an important role in the regulation of many mental states and the processing of emotions in the central nervous system. Serotonergic neurons in the central nervous system, including the dorsal raphe (DR) and median raphe (MR) nuclei, are spatially clustered in the brainstem and provide ascending innervation to the entire forebrain and midbrain. Both between and within the DR and MR, these serotonergic neurons have different cellular characteristics, developmental origin, connectivity, physiology, and related behavioral functions. Recently, an understanding of the heterogeneity of the DR and MR serotonergic neurons has been developed at the molecular level. In birds, emotion-related behavior is suggested to be modulated by the 5-HT system. However, correspondence between the raphe nuclei of birds and mammals, as well as the cellular heterogeneity in the serotonergic neurons of birds are poorly understood. To further understand the heterogeneity of serotonergic neurons in birds, we performed a molecular dissection of the chick brainstem using in situ hybridization. In this study, we prepared RNA probes for chick orthologs of the following serotonin receptor genes: 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1E, 5-HTR1F, 5-HTR2A, 5-HTR2B, 5-HTR2C, 5-HTR3A, 5-HTR4, 5-HTR5A, and 5-HTR7. We showed that the expression pattern of 5-HT receptors in the serotonin neurons of chick DR and MR may vary, suggesting heterogeneity among and within the serotonin neurons of the DR and MR in the chick brainstem. Our findings regarding the molecular properties of serotonergic neurons in the bird raphe system will facilitate a good understanding of the correspondence between bird and mammalian raphes.

The dorsal arcopallium of chicks displays the expression of orthologs of mammalian fear related serotonin receptor subfamily genes.

Fear is an adaptive emotion that elicits defensive behavioural responses against aversive threats in animals. In mammals, serotonin receptors (5-HTRs) have been shown to modulate fear-related neural circuits in the basolateral amygdala complex (BLA). To understand the phylogenetic continuity of the neural basis for fear, it is important to identify the neural circuit that processes fear in other animals. In birds, fear-related behaviours were suggested to be processed in the arcopallium/amygdala complex and modulated by the serotonin (5-HT) system. However, details about the distribution of 5-HTRs in the avian brain are very sparsely reported, and the 5-HTR that is potentially involved in fear-related behaviour has not been elucidated. In this study, we showed that orthologs of mammalian 5-HTR genes that are expressed in the BLA, namely 5-HTR1A, 5-HTR1B, 5-HTR2A, 5-HTR2C, 5-HTR3A, and 5-HTR4, are expressed in a part of the chick arcopallium/amygdala complex called the dorsal arcopallium. This suggests that serotonergic regulation in the dorsal arcopallium may play an important role in regulating fear-related behaviour in birds. Our findings can be used as a basis for comparing the processing of fear and its serotonergic modulation in the mammalian amygdala complex and avian arcopallium/amygdala complex.

Blockade of muscarinic acetylcholine receptor by scopolamine impairs the memory formation of filial imprinting in domestic chicks (Gallus Gallus domesticus).

Filial imprinting in precocial birds is a useful model for studying memory formation in early learning. The intermediate medial mesopallium (IMM) in the dorsal telencephalon is one of the critical brain regions where the releases of several neurotransmitters increase after the start of imprinting training. Among the increased neurotransmitters, the role of acetylcholine in imprinting has remained unclear. Acetylcholine in the mammalian brain plays an important role in encoding new memories. The muscarinic acetylcholine receptor subtype 1 (M1 receptor) and subtype 3 (M3 receptor) in the hippocampus and cortex of mammalian brain have been shown to be necessary for memory encoding. In this study, we examined whether the imprinting acquisition in chick can be impaired by injecting muscarinic acetylcholine receptor (mAChR) antagonist scopolamine into the bilateral IMM. We show that the injection of scopolamine decreased the preference for the imprinting object in the test, but did not affect the number of approaches to the imprinting object during training. Immunoblotting and immunohistochemistry revealed that M3 receptors were expressed in the IMM. Our data suggest that acetylcholine is involved in the memory formation of imprinting through M3 receptors in the IMM. The scopolamine-injected chicks may be useful as an animal model for dementia such as Alzheimer's disease.

Combined predisposed preferences for colour and biological motion make robust development of social attachment through imprinting.

To study how predisposed preferences shape the formation of social attachment through imprinting, newly hatched domestic chicks (Gallus gallus domesticus) were simultaneously exposed to two animations composed of comparable light points in different colours (red and yellow), one for a walking motion and another for a linear motion. When a walking animation in red was combined with a linear one in yellow, chicks formed a learned preference for the former that represented biological motion (BM). When the motion-colour association was swapped, chicks failed to form a preference for a walking in yellow, indicating a bias to a specific association of motion and colour. Accordingly, experiments using realistic walking chicken videos revealed a preference for a red video over a yellow one, when the whole body or the head was coloured. On the other hand, when the BM preference had been pre-induced using an artefact moving rigidly (non-BM), a clear preference for a yellow walking animation emerged after training by the swapped association. Even if the first-seen moving object was a nonbiological artefact such as the toy, the visual experience would induce a predisposed BM preference, making chicks selectively memorize the object with natural features. Imprinting causes a rapid inflow of thyroid hormone in the telencephalon leading to the induction of the BM preference, which would make the robust formation of social attachment selectively to the BM-associated object such as the mother hen.

Complex social ecology needs complex machineries of foraging.

Uncertainty is caused not only by environmental changes, but also by social interference resulting from competition over food resources. Actually, foraging effort is socially facilitated, which, however, does not require incentive control by the dopamine system; Zajonc's "drive" theory is thus questionable. Instead, social adjustments may be pre-embedded in the limbic network responsible for decisions of appropriate effort-cost investment.

The chick pallium displays divergent expression patterns of chick orthologues of mammalian neocortical deep layer-specific genes.

The avian pallium is organised into clusters of neurons and does not have layered structures such as those seen in the mammalian neocortex. The evolutionary relationship between sub-regions of avian pallium and layers of mammalian neocortex remains unclear. One hypothesis, based on the similarities in neural connections of the motor output neurons that project to sub-pallial targets, proposed the cell-type homology between brainstem projection neurons in neocortex layers 5 or 6 (L5/6) and those in the avian arcopallium. Recent studies have suggested that gene expression patterns are associated with neural connection patterns, which supports the cell-type homology hypothesis. However, a limited number of genes were used in these studies. Here, we showed that chick orthologues of mammalian L5/6-specific genes, nuclear receptor subfamily 4 group A member 2 and connective tissue growth factor, were strongly expressed in the arcopallium. However, other chick orthologues of L5/6-specific genes were primarily expressed in regions other than the arcopallium. Our results do not fully support the cell-type homology hypothesis. This suggests that the cell types of brainstem projection neurons are not conserved between the avian arcopallium and the mammalian neocortex L5/6. Our findings may help understand the evolution of pallium between birds and mammals.

Wnt-2b in the intermediate hyperpallium apicale of the telencephalon is critical for the thyroid hormone-mediated opening of the sensitive period for filial imprinting in domestic chicks (Gallus gallus domesticus).

Filial imprinting is the behavior observed in chicks during the sensitive or critical period of the first 2-3 days after hatching; however, after this period they cannot be imprinted when raised in darkness. Our previous study showed that temporal augmentation of the endogenous thyroid hormone 3,5,3'-triiodothyronine (T3) in the telencephalon, by imprinting training, starts the sensitive period just after hatching. Intravenous injection of T3 enables imprinting of chicks on days 4 or 6 post-hatching, even when the sensitive period has ended. However, the molecular mechanism of how T3 acts as a determinant of the sensitive period is unknown. Here, we show that Wnt-2b mRNA level is increased in the T3-injected telencephalon of 4-day old chicks. Pharmacological inhibition of Wnt signaling in the intermediate hyperpallium apicale (IMHA), which is the caudal area of the telencephalon, blocked the recovery of the sensitive period following T3 injection. In addition, injection of recombinant Wnt-2b protein into the IMHA helped chicks recover the sensitive period without the injection of T3. Lastly, we showed Wnt signaling to be involved in imprinting via the IMHA region on day 1 during the sensitive period. These results indicate that Wnt signaling plays a critical role in the opening of the sensitive period downstream of T3.

Gene expression of Dio2 (thyroid hormone converting enzyme) in telencephalon is linked with predisposed biological motion preference in domestic chicks.

Filial imprinting leads to the formation of social attachment if training is performed during a brief sensitive period after hatching. We found that thyroid hormone (3,5,3'-triiodothyronine, T3) acts as a critical determining factor of the sensitive period in domestic chicks. Imprinting upregulates gene expression of the converting enzyme (Dio2, type 2 iodothyronine deiodinase) in the telencephalon, leading to increased brain T3 content. If systemically applied, T3 facilitates imprinting in aged chicks even after the sensitive period is over. Imprinting is also associated with the rapid development of visual perception. Exposure to motion pictures induces a predisposed preference to Johansson's biological motion (BM), and those individuals with higher BM preference are more easily imprinted. Here, we examined whether Dio2 expression is also linked with BM predisposition. Chicks were trained by a rotating red block, and tested for imprinting (experiment 1) and BM preference (experiment 2). To examine the time courses of behavioural and physiological processes, Dio2 expression in telencephalon was compared among three groups: naïve control chicks, and chicks trained for a short (0.5 h) or long period (2 h). In experiment 1, higher Dio2 expression appeared in the 2-h group than in the 0.5-h/control groups, but it was not correlated with the individual imprinting score. In experiment 2, a significant positive correlation appeared between Dio2 expression and BM preference in 2-h-trained chicks. Memory priming by T3 is therefore functionally linked to BM preference induction, leading to successful imprinting to natural objects even when they are initially exposed to artificial objects.