Serotonin Imprinting in Chicken Embryonic Development

Xiaohong Huang, Purdue University


Hormonal imprinting occurs immediately at the first encounter between a hormone and its receptor-signal transduction system. It causes changes in brain development, including alterations in the biosynthesis of hormones and the sensitivity of relative receptors, affecting the animal’s physiological homeostasis and behavioral exhibition. Serotonin (5-HT) and its receptors appear early during prenatal development. 5-HT, acting as a neurotrophic factor, influences brain development, and, acting as a neurotransmitter, regulates multiple biological functions. For example, 5-HT interacts with dopamine (DA) signaling in regulating animal aggression. It has been reported that aggressive behaviors are reduced in 5-HT (injected 10 ug/egg before incubation) treated pullets at 9wk and 18wk compared to the saline controls. We hypothesized that excessive 5-HT administration immediately before egg incubation (at the stage of primary hypoblast) would imprint the 5-HT receptors, enhancing the receptor sensitivity to 5-HT and serotonergic system functions. Moreover, we expected that the imprinting effects would be amplified during brain development via the interactions between the 5-HTergic system and other neuronal systems, such as the dopaminergic (DAergic) system. To examine the hypothesis, fertilized eggs from a white egg laying strain were used. Two trials were conducted to study 1) the natural maturation process of the 5-HTergic system and its effects on the development of DAergic system and 2) 5-HT imprinting effects on the development of the 5-HTergic/DAergic system during mid-late embryogenesis. ^ Trial 1. The brain samples were collected from intact embryos at embryonic day (E) 12, 14, 16, 19 and 20. Morphological, molecular and metabolic analyses indicated that neuronal growth was the main trend during E12-19, while a remarkable regression of the 5-HTergic neurons was revealed during E14-16, and the 5-HTergic system reached relatively maturity before hatching. The metabolic activities and packing densities of the midbrain and hypothalamic DAergic cell groups were increased during E16-19. The unique developing time sequence between the 5-HTergic and DAergic systems confirmed that the 5-HTergic system plays an important role in regulating the prenatal development of the DAergic system. ^ Trial 2. The eggs were randomly divided into three groups: control (injected with saline) and experimental 1 and 2 (injected with 5-HT, 10 and 20 ug/egg, respectively). The brain samples were mainly collected at E14 and E19. At E14, the effects of 5-HT treatment on the development of the 5-HTergic system were detected in both 10 ug and 20 ug groups by reprogramming the 5-HTergic system developmental process. The 10 ug and 20 ug 5-HT imprinting advanced the appearance of 5-HTergic nuclei and the raphe neuronal regression. At E14, the 20 ug but not 10 ug 5-HT treatment upregulated the raphe 5-HT concentration without affecting the metabolic activity of the DAergic system. In addition, the Pet-1/Htr1a autoregulatory loop functioned differently between the 10 ug and 20 ug groups from E14. The 10 ug group had higher Htr1a expression but lower 5-HT level; while the 20 ug group had lower Htr1a expression but higher 5-HT concentration, which may be associated with the different degrees of dendritic regression, leading to the greater reduction of Htr1a binding sites in the 20 ug group. At E19, the differences in the raphe 5-HT concentrations and Htr1a expression among three groups were inherited and amplified from the mid-embryonic stage to the pre-hatching stage. In the pons, the availability of synaptic 5-HT was increased in both 10 ug and 20 ug groups via different paths, which suggested the increases in 5-HTergic terminal activities on the midbrain DAergic groups. The 5-HT imprinting in the 10 ug group facilitated the release of 5-HT at the synaptic terminals via increased the 5-HTT reuptake activity and reduced 5-HT degradation (a low 5-HIAA/5-HT ratio); while in the 20 ug group, it increased the synaptic 5-HT concentrations via enhanced raphe 5-HT neurons’ synthetic function. The hypothalamic 5-HT metabolisms were significantly decreased by the 10 ug and 20 ug 5-HT imprinting, and the 5-HTT reuptake activities and 5-HTergic axons that terminated at the ventromedial hypothalamus were increased in both 10 ug and 20 ug groups, which suggested increases in 5-HTergic terminal activities in the hypothalamus. Hence, both 10 ug and 20 ug 5-HT imprinting facilitated the neuronal development of both midbrain and hypothalamic DAergic groups, such as the ventral tegmental area and periventricular nucleus, via the increased synaptic 5-HT availabilities. ^ The previous study in our lab has shown that 5-HT imprinting reduces postnatal aggressive behavior with increased 5-HT proportion in the 5-HT/DA balance. Its mechanisms were further detected in the current study. The elevated synaptic 5-HT availabilities by both 10 ug and 20 ug 5-HT imprinting may have reprogrammed the development of the HPA axis during embryogenesis, mediate its activity during stress responses, and increase 5-HTergic dominance in regulating 5-HT/DA balance during postnatal life. Through these changes, 5-HT imprinting regulates chicken physiological homeostasis and behavioral exhibition, such as inhibiting aggression. The results reveal that 5-HT imprinting could be a useful tool to improve laying hen health and welfare via prenatally reprogramming neural circuitry.^




Heng-wei Cheng, Purdue University, Shihuan Kuang, Purdue University.

Subject Area

Neurosciences|Developmental biology

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