High-dose maternal folic acid supplementation before conception impairs reversal learning in offspring mice

Abstract

Maternal folic acid (FA) supplementation prior to and during gestation is recommended for the prevention of neural tube closure defects in the developing embryo. Prior studies, however, suggested that excessive FA supplementation during gestation can be associated with toxic effects on the developing organism. Here, we address whether maternal dietary folic acid supplementation at 40 mg/kg chow (FD), restricted to a period prior to conception, affects neurobehavioural development in the offspring generation. Detailed behavioural analyses showed reversal learning impairments in the Morris water maze in offspring derived from dams exposed to FD prior to conceiving. Furthermore, offspring of FD dams showed minor and transient gene expression differences relative to controls. Our data suggest that temporary exposure of female germ cells to FD is sufficient to cause impaired cognitive flexibility in the subsequent generation.

Figure 1

Schematic illustration of the study design.

Figure 2

Acquisition and reversal training in the Morris water maze. Visual representations of swim tracks are shown for each day and group. Relative occupancy values during training and reversal training in the Morris water maze are indicated by the colour code (see colour bar; red denotes high occupancy, blue denotes low occupancy values). The yellow circle denotes the platform location during training and reversal training. TQ = target quadrant, AL = adjacent left, AR = adjacent right, OQ = opposite quadrant, OTQ = old target quadrant, NTQ = new target quadrant. CD = F1 offspring of control dams. FD = F1 offspring of dams that received the folic acid-supplemented diet prior to mating. Data are presented as mean ± S.E.M. (CD: n = 24, FD: n = 20).

Figure 3

Morris water maze probe trial data. The figure shows Morris water maze probe trial data regarding probe trials delivered on days 5, 7 and 10. (A) Cumulative swim track representations (heat maps) are shown for both groups and for each day. Relative occupancy values during the probe trials in the Morris water maze are indicated by the colour code (see colour bar; red denotes high occupancy, blue denotes low occupancy values). (B–D) Quadrant occupancy during probe trials on day 5 (B), day 7 (C) and during the reversal probe trial on day 10 (D). (E–G) Average distance to the target location (average proximity) during probe trials on day 5 (E), day 7 (F) and during the reversal probe trial on day 10 (G). TQ = target quadrant, AL = adjacent left, AR = adjacent right, OQ = opposite quadrant, OTQ = old target quadrant, NTQ = new target quadrant (former OQ). CD = F1 offspring of control dams. FD = F1 offspring of dams that received the folic acid-supplemented diet prior to mating. Data are presented as mean ± S.E.M; asterisks denote significant differences (**p < 0.01, ***p < 0.001; CD: n = 24, FD: n = 20).

Figure 4

Additional behavioural data and analyses. (A) Mean velocity during probe trials in the Morris water maze. (B) Distance moved during a 20 min open field test. (C) Percentage of time spent freezing during baseline, the post-shock period and during the context test given one day after training in a contextual fear conditioning paradigm. (D) Exploration times (known object, novel object) in the context of an object place recognition test. (E) Latency to fall off an accelerating rotarod. (F) Body weight (at 12 weeks of age). CD = F1 offspring of control dams. FD = F1 offspring of dams that received the folic acid-supplemented diet prior to mating. Data are presented as mean ± S.E.M.; asterisks denote significant differences (*p < 0.05, **p < 0.01, ***p < 0.001; CD: n = 24, FD: n = 20).

Figure 5

Weighted correlation network analysis (WGCNA) of hippocampal expression data in CD and FD F1 mice. (A) Graphs show module-trait relationships and corresponding p-values (presented as –log of the p-value) of the top ten modules detected via WGCNA (module names are provided on the x-axes). The p-value threshold of –log (0.05) is indicated by a dotted line. (B) Scatterplot of gene significance (GS.group) versus module membership (kME) for the darkgrey module. We observed a strong correlation between GS values for group (maternal diet) and module membership. (C) The graph shows the top enriched canonical pathways among genes with membership in the darkgrey module.

Figure 6

Expression changes of candidate genes in hippocampi of CD and FD F1 mice evaluated by qPCR. The graph shows normalized fold change values relative to control samples. Data are presented as mean ± S.E.M.; asterisks denote significant differences (*p < 0.05; n = 6 per group).