Journal: Nutrients

Article Title: Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism

doi: 10.3390/nu12061716

Figure Lengend Snippet: Open field (OF) test in 3-week-old male and female offspring. ( A ) FASD pups traveled greater distances than CD pups (left panel). Representative tracking plots for a CD and a FASD animal are shown (right panel). ( B ) FASD pups traveled at higher mean speeds than CD pups. ( C ) FASD pups spent more time grooming than CD pups. ( D ) FASD pups spent more time rearing than CD pups. ( E ) FASD pups had more rearing episodes than CD pups, although the difference did not reach statistical significance. ( F ) FASD pups spent more total time active (i.e., grooming, rearing and traveling) than CD pups. ( G ) There were no differences in the anxiety index. Anxiety index is calculated as distance traveled in the 4 center squares divided by total distance traveled. n = 15–17/group, 10–11 litters/diet. White bars: CD animals, gray bars: FASD animals. Values are means ± SEM. p values from linear mixed-model analysis (including maternal diet and offspring sex as fixed factors and litter as a random factor) are indicated at the top of each graph. CD: Control diet, FASD: Folic acid supplemented diet, D: Diet, S: Sex, D×S: Diet × Sex interaction.

Article Snippet: At weaning, female C57BL/6 mice were randomly placed on amino acid defined Control Diet (CD) (2 mg FA/kg diet, recommended level for rodents) [ ] (TD.01369, Harlan Laboratories, Inc., Madison, WI, USA) or Folic Acid Supplemented Diet (FASD), which contained five times the amount of FA recommended for rodents (10 mg FA/kg diet, TD.08278, Harlan Laboratories, Inc., Madison, WI, USA) ( ).

Techniques: Control

Journal: Nutrients

Article Title: Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism

doi: 10.3390/nu12061716

Figure Lengend Snippet: NOR test in 3-week-old male and female offspring. ( A ) CD pups spent more time exploring the novel object compared to the familiar object, whereas FASD pups spent the same or less time exploring the novel object compared to the familiar object. ( B ) Short-term memory impairment, indicated by the negative DI, was observed due to FASD during the NOR test. A negative DI (calculated as time spent exploring novel object minus time spent exploring familiar object divided by total time exploring both novel and familiar objects) indicates that less time was spent with the novel object compared to the familiar object. White bars: CD animals, gray bars: FASD animals. ( C ) DI negatively correlated with total activity time in the OF test. n = 10–13/group, 9–11 litters/diet. Values are means ± SEM. p values from linear mixed-model analysis (including maternal diet and offspring sex as fixed factors and litter as a random factor) are indicated at the top of each graph. * p < 0.05, *** p < 0.001 ( t -test). CD: Control diet, FASD: Folic acid supplemented diet, D: Diet, S: Sex, D×S: Diet × Sex interaction, r : Pearson’s correlation coefficient.

Article Snippet: At weaning, female C57BL/6 mice were randomly placed on amino acid defined Control Diet (CD) (2 mg FA/kg diet, recommended level for rodents) [ ] (TD.01369, Harlan Laboratories, Inc., Madison, WI, USA) or Folic Acid Supplemented Diet (FASD), which contained five times the amount of FA recommended for rodents (10 mg FA/kg diet, TD.08278, Harlan Laboratories, Inc., Madison, WI, USA) ( ).

Techniques: Activity Assay, Control

Journal: Nutrients

Article Title: Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism

doi: 10.3390/nu12061716

Figure Lengend Snippet: MTHFR protein in maternal and offspring liver. ( A ) Total immunoreactive MTHFR protein (normalized to vinculin expression) was reduced in livers of FASD mothers ( n = 9–10/group). ( B ) The ratio of the phosphorylated isoform (less active) to the non-phosphorylated isoform was increased in FASD mothers ( n = 10/group). ( C ) Representative Western blot of liver extracts from CD and FASD mothers. ( D ) Total immunoreactive MTHFR protein (normalized to vinculin expression) was reduced in livers of FASD pups compared to CD pups, and in female pups compared to male pups ( n = 5–7/group). ( E ) The ratio of the phosphorylated isoform to the non-phosphorylated isoform was increased in FASD male pups ( n = 5–7/group). ( F ) Representative Western blot of liver extracts from 4-week-old male and female pups. White bars: CD animals, gray bars: FASD animals. Values are means ± SEM. In graphs ( A , B ), asterisks correspond to t -test associated p values, * p < 0.05, *** p < 0.001. In graphs ( C , D ), p values from 2-factor ANOVA are indicated at the top of each graph, and Tukey post-hoc significant p values are indicated as ++ p < 0.01. CD: Control diet, FASD: Folic acid supplemented diet, D: Diet, S: Sex, D×S: Diet × Sex interaction, P-MTHFR, phosphorylated MTHFR isoform.

Article Snippet: At weaning, female C57BL/6 mice were randomly placed on amino acid defined Control Diet (CD) (2 mg FA/kg diet, recommended level for rodents) [ ] (TD.01369, Harlan Laboratories, Inc., Madison, WI, USA) or Folic Acid Supplemented Diet (FASD), which contained five times the amount of FA recommended for rodents (10 mg FA/kg diet, TD.08278, Harlan Laboratories, Inc., Madison, WI, USA) ( ).

Techniques: Expressing, Western Blot, Control

Journal: Nutrients

Article Title: Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism

doi: 10.3390/nu12061716

Figure Lengend Snippet: Concentrations of choline-derived metabolites measured by LC-MS in offspring liver. ( A ) FASD pups showed reduced betaine compared to CD pups. ( B ) FASD pups showed increased SAM compared to CD pups, and male pups showed reduced SAM compared to females. ( C ) Male pups showed reduced choline compared to females. ( D ) Male pups showed reduced PCho compared to females. n = 7–8/group. White bars: CD animals, gray bars: FASD animals. Values represent means ± SEM. p values from 2-factor ANOVA are indicated at the top of each graph, and Tukey post-hoc significant p values are indicated as + p < 0.05, ++ p < 0.01. CD: Control diet, FASD: Folic acid supplemented diet, D: Diet, S: Sex, D×S: Diet × Sex interaction.

Article Snippet: At weaning, female C57BL/6 mice were randomly placed on amino acid defined Control Diet (CD) (2 mg FA/kg diet, recommended level for rodents) [ ] (TD.01369, Harlan Laboratories, Inc., Madison, WI, USA) or Folic Acid Supplemented Diet (FASD), which contained five times the amount of FA recommended for rodents (10 mg FA/kg diet, TD.08278, Harlan Laboratories, Inc., Madison, WI, USA) ( ).

Techniques: Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Control

Journal: Nutrients

Article Title: Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism

doi: 10.3390/nu12061716

Figure Lengend Snippet: Concentrations of choline-derived metabolites measured by LC-MS in offspring cerebral cortex. ( A ) FASD male pups showed a reduction in GPC compared to CD male pups; females did not show dietary changes. GPC concentration in male pups positively correlated with the DI in the NOR test; female pups did not show a significant correlation. ( B ) FASD male pups showed a reduction in PtdCho compared to CD males; females did not show dietary changes. PtdCho levels in male pups positively correlated with the DI in the NOR test; female pups did not. ( C ) FASD male pups showed a reduction in SM compared to CD males; females did not. SM concentration in male pups positively correlated with the DI in NOR test; female pups did not show this correlation. ( D ) Acho concentrations in female pups positively correlated with the DI in NOR test; male pups did not show this correlation. n = 7–8/group. White bars: CD animals, gray bars: FASD animals. Bar graphs represent means ± SEM. p values from 2-factor ANOVA are indicated at the top of each graph. Asterisks correspond to t test associated p values, indicated as * p < 0.05, ** p < 0.01. CD: Control diet, FASD: Folic acid supplemented diet, D: Diet, S: Sex, D×S: Diet × Sex interaction, r : Pearson’s correlation coefficient.

Article Snippet: At weaning, female C57BL/6 mice were randomly placed on amino acid defined Control Diet (CD) (2 mg FA/kg diet, recommended level for rodents) [ ] (TD.01369, Harlan Laboratories, Inc., Madison, WI, USA) or Folic Acid Supplemented Diet (FASD), which contained five times the amount of FA recommended for rodents (10 mg FA/kg diet, TD.08278, Harlan Laboratories, Inc., Madison, WI, USA) ( ).

Techniques: Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Control

Journal: Nutrients

Article Title: Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism

doi: 10.3390/nu12061716

Figure Lengend Snippet: PtdCho and SM measured by MALDI-IMS in offspring brain. ( A ) The two most abundant species of PtdCho (m/z 734 + 760) were decreased in neocortex of FASD pups. ( B ) These 2 species were also decreased in hippocampus of FASD pups ( C ) Representative mapping of the different signal intensities (arbitrary units) for PtdCho (m/z 734) in neocortex (designated by a blue line), hippocampus (yellow line), and cerebellum (red line). ( D ) SM was decreased due to FASD in neocortex, predominantly in males. ( E ) SM was decreased due to FASD in hippocampus although this change was not statistically significant. ( F ) Representative mapping of the different signal intensities (arbitrary units) for SM in neocortex (designated by a blue line), hippocampus (yellow line), and cerebellum (red line). n = 4/group. White bars: CD animals, gray bars: FASD animals. Bar graphs represent means ± SEM. p values from 2-factor ANOVA are indicated at the top of each graph. Asterisks correspond to Tukey post-hoc p values, indicated as (+) p = 0.064, + p < 0.05, ++ p < 0.01. CD: Control diet, FASD: Folic acid supplemented diet, D: Diet, S: Sex, D×S: Diet × Sex interaction.

Article Snippet: At weaning, female C57BL/6 mice were randomly placed on amino acid defined Control Diet (CD) (2 mg FA/kg diet, recommended level for rodents) [ ] (TD.01369, Harlan Laboratories, Inc., Madison, WI, USA) or Folic Acid Supplemented Diet (FASD), which contained five times the amount of FA recommended for rodents (10 mg FA/kg diet, TD.08278, Harlan Laboratories, Inc., Madison, WI, USA) ( ).

Techniques: Control