Table_4_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.

在孕育期补充叶酸（FA）以预防神经管缺陷及其他出生缺陷的建议已被广泛采纳。经过二十年的强制在食品中添加叶酸，北美人群血清中叶酸和未代谢的叶酸浓度显著上升。然而，关于过量摄入叶酸是否会损害神经发育和行为，尚存在争议。在本研究中，我们于交配前一周以及整个妊娠和哺乳期间，对小鼠施以约2.5倍（叶酸中等剂量，MFA）或10倍（叶酸高剂量，HFA）的饮食需求量的叶酸，并利用开放式场测试、三室社交性和社交新奇性测试、倾斜迷宫、旋转杆和莫里斯水迷宫等方法，考察了成年雄性后代的各项行为。我们发现，早期生命阶段的MFA补充导致成年期长期体重增加、焦虑样行为增强，以及社交偏好、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测序显示，叶酸补充改变了断奶时大脑基因的表达，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。定量实时荧光定量PCR（qRT-PCR）和蛋白质印迹分析证实了这些基因表达的增加。我们的研究结果表明，早期生命阶段的叶酸补充会影响断奶小鼠的基因表达，并以剂量敏感的方式在成年行为上展现出长期的影响。