Table_5_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.

围孕期叶酸（folic acid, FA）补充被推荐用于预防神经管缺陷及其他出生缺陷。在强制推行叶酸强化食品政策20年后，北美人群的血清叶酸及未代谢叶酸浓度显著升高，但过量摄入叶酸是否会损害神经发育与行为，目前仍存在争议。本研究在小鼠交配前1周至整个妊娠及哺乳期，为其喂食相当于膳食推荐摄入量2.5倍（中等剂量叶酸，MFA）或10倍（高剂量叶酸，HFA）的叶酸，并通过旷场试验、三箱社交与社交新颖性试验、高架十字迷宫、旋转棒实验以及莫里斯水迷宫，检测成年雄性子代的行为表现。研究发现，生命早期补充中等剂量叶酸会使成年小鼠的长期体重增长增加，提升焦虑样行为，并损害社交偏好、运动学习与空间学习能力，但未改变运动能力与空间记忆。与之相反，高剂量叶酸补充仅会引发轻微的行为异常。RNA测序结果显示，叶酸补充会改变断奶小鼠的脑部基因表达，其中与对照组及高剂量叶酸组相比，中等剂量叶酸组小鼠的Fos及其相关基因表达显著上调。实时荧光定量PCR（quantitative real time-PCR, qRT-PCR）与蛋白质印迹实验证实了这些基因的表达上调。本研究结果表明，生命早期补充叶酸会改变断奶小鼠的脑部基因表达，并以剂量依赖的方式对成年小鼠的行为造成长期损害。