Effects of in ovo injection of serotonin on behavior and hypothalamic genes expression in post hatch-chicks

Abstract

Serotonin (5-HT) is essential for neuronal development and behavioral regulation. Serotonin, upon in ovo administration, has been previously reported to modulate aggressive behavior in avian species, however it still remains unknown if maternal serotonin affects chick behavior through hypothalamic gene expression. We injected an equal volume of saline (control) and either a 5 μg (low) or a 15 μg (high) dose of 5-HT into embryos on embryonic day 11 (E11). The blood concentration of 5-HT was determined in chicks on post-hatching D3 (Day 3) and D45. The behavioral fear and aggression were analyzed at D10 and D52. In isolation test, the latency to vocalize and walk, the duration of vocalizations and walking were recorded. Hypothalamic expression of genes related to the serotonin pathway and the methylation status of hypothalamic 5-HT 1A receptor (5-HTR1A) were measured at D56. Chicks treated with embryonic 5-HT exhibited a certain decrease in the degree of fear as determined by the duration of vocalizations (p < 0.05) in an isolation test. They also exhibited a significantly less aggressive behavior (p < 0.05) as compared to chicks given saline control. We also noticed that the higher dosage was consequently associated with elevated concentration of 5-HT in blood. Exposure to high dosage of 5-HT significantly down-regulated Monoamine oxidase A (MAO-A) and B (MAO-B). Alternatively, the mRNA expression of Hypothalamic tryptophan hydroxylase 2 (TPH2) was significantly up-regulated (p < 0.05). However, the mRNA levels of Tryptophan hydroxylase 1 (TPH1) and the transporter for 5-HT (5-HTT) remained unchanged. Interestingly, 5-HTR1A was found to be significantly increased in the high-dosage group (p < 0.05). The promoter region of 5-HTR1A gene was significantly hypomethylated (p < 0.05) which correlated negatively with the mRNA expression of 5-HTR1A genes (r=-0.682, p = 0.005). These results indicate that in ovo serotonin injection affects aggressive and fearful behaviors by modifying the expression of genes involved in the serotonergic pathway, through DNA methylation mediated epigenetic mechanisms.