Table_6_Excess Folic Acid Supplementation Before and During Pregnancy and Lactation Activates Fos Gene Expression and Alters Behaviors in Male Mouse Offspring.XLS

Periconceptional folic acid (FA) supplementation is recommended to prevent neural tube defects and other birth defects. After 20 years mandate food fortification with FA, serum concentration of folate and unmetabolized FA increased significantly in the North American population. But whether excess FA intake impairs neurodevelopment and behavior is still controversial. Here, we treated mice with approximately 2.5-fold (moderate dose of FA, MFA) or 10-fold (high dose of FA, HFA) the dietary requirement of FA 1 week before mating and throughout pregnancy and lactation, and examined behaviors in adult male offspring using open field test, three-chamber sociability and social novelty test, elevated plus maze, rotarod and Morris water maze. We found that early life MFA supplementation increased long-term body weight gain in adults, elevated anxiety-like behavior, and impaired social preference, motor learning and spatial learning ability without modifying motor ability and spatial memory. In contrast, HFA supplementation only induced mild behavioral abnormality. RNA sequencing revealed that FA supplementation altered the expression of brain genes at weaning, among which Fos and related genes were significantly up-regulated in MFA mice compared with control and HFA mice. Quantitative real time-PCR (qRT-PCR) and western blots confirmed the increase of these genes. Our results suggested that FA supplementation during early life stage affected gene expression in weaning mice, and exhibited long-term impairments in adult behaviors in a dose-sensitive manner.

围孕期补充叶酸（FA）旨在预防神经管缺陷及其他出生缺陷。自20年前实施FA强化食品政策以来，北美人群血清中的叶酸和未代谢的FA浓度显著上升。然而，关于过量摄入FA是否会损害神经发育和行为，至今仍存在争议。本研究中，我们在交配前一周及整个孕期和哺乳期，将小鼠分别给予约为膳食需求量2.5倍（中等剂量FA，MFA）或10倍（高剂量FA，HFA）的FA，并通过开放式场测试、三室社交性和社交新奇性测试、倾斜加法迷宫、旋转杆和莫里斯水迷宫等方法，检测了成年雄性后代的行为。我们发现，早期生命阶段的MFA补充导致成年后长期体重增加、焦虑样行为升高、社交偏好、运动学习和空间学习能力受损，而未改变运动能力和空间记忆。相比之下，HFA补充仅引起轻微的行为异常。RNA测序结果显示，FA补充改变了断奶时脑基因的表达，其中与Fos相关的基因在MFA小鼠中相对于对照组和HFA小鼠显著上调。定量实时PCR（qRT-PCR）和蛋白质印迹分析证实了这些基因表达的升高。我们的研究结果提示，早期生命阶段的FA补充会影响断奶小鼠的基因表达，并以剂量敏感的方式导致成年行为的长期损害。