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.

Serotonin (5-hydroxytryptamine, 5-HT) is a phylogenetically conserved modulator of numerous aspects of neural functions. Serotonergic neurons in the dorsal and median raphe nucleus provide ascending innervation to the entire forebrain and midbrain. Another important neural modulatory system exists in the midbrain, the dopaminergic system, which is associated to reward processing and motivation control. Dopaminergic neurons are distributed and clustered in the brain, classically designated as groups A8–A16. Among them, groups A8–A10 associated with reward processing and motivation control are located in the midbrain and projected to the forebrain. Recently, midbrain dopaminergic neurons were shown to be innervated by serotonergic neurons and modulated by 5-HT, with the crosstalk between serotonergic and dopaminergic systems attracting increased attention. In birds, previous studies revealed that midbrain dopaminergic neurons are located in the A8-A10 homologous clusters. However, the detailed distribution of dopaminergic neurons and the crosstalk between serotonergic and dopaminergic systems in the bird are poorly understood. To improve the understanding of the regulation of the dopaminergic by the serotonergic system, we performed in situ hybridization in the chick brainstem. We prepared RNA probes for chick orthologues of dopaminergic neuron-related genes; tyrosine hydroxylase (TH) and dopa decarboxylase (DDC), noradrenaline related genes; noradrenaline transporter (NAT) and dopamine beta-hydroxylase (DBH), and 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 confirmed that the expression of tyrosine hydroxylase (TH) and NAT was well matched in all chick dopaminergic nuclei examined. This supported that the compensation of the function of dopamine transporter (DAT) by NAT is a general property of avian dopaminergic neurons. Furthermore, we showed that 5-HTR1A and 5-HTR1B were expressed in midbrain dopaminergic nuclei, suggesting the serotonergic regulation of the dopaminergic system via these receptors in chicks. Our findings will help us understand the interactions between the dopaminergic and serotonergic systems in birds at the molecular level.

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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.