Brain Susceptibility to Methyl Donor Deficiency: From Fetal Programming to Aging Outcome in Rats

Abstract

Deficiencies in methyl donors, folate, and vitamin B12 are known to lead to brain function defects. Fetal development is the most studied but data are also available for such an impact in elderly rats. To compare the functional consequences of nutritional deficiency in young versus adult rats, we monitored behavioral outcomes of cerebellum and hippocampus circuits in the offspring of deficient mother rats and in adult rats fed a deficient diet from 2 to 8 months-of-age. We present data showing that the main deleterious consequences are found in young ages compared to adult ones, in terms of movement coordination and learning abilities. Moreover, we obtained sex and age differences in the deleterious effects on these functions and on neuronal layer integrity in growing young rats, while deficient adults presented only slight functional alterations without tissue damage. Actually, the cerebellum and the hippocampus develop and maturate according to different time lap windows and we demonstrate that a switch to a normal diet can only rescue circuits that present a long permissive window of time, such as the cerebellum, whereas the hippocampus does not. Thus, we argue, as others have, for supplements or fortifications given over a longer time than the developmental period.

Keywords: brain development; fetal programming; functional deficits; long-term effects; neuroplasticity.

Figure 1

Behavioral monitoring of pups at the end of the deficient period (CM: control males; DM: deficient males; CF: control females; DF: deficient females). (A,B) Performances in the linear walking test to join the home-cage at PN 18. (C–F) Behavioral characteristics presented in the elevated plus maze at PN 20. * p < 0.05; ** p < 0.01; *** p < 0.001; n = 11 to 28 (see table F).

Figure 2

Biochemical analysis of plasma and the evaluation of the general activity at the end of the deficient period. (A) Plasma concentration of Vitamin B12, Vitamin B9, and homocysteine. (B,C) evaluation of locomotion and exploration levels at PN 21. *** p < 0.001; n = 11 to 28.

Figure 3

Evaluation of the motor and coordination functions after the switch to a normal diet at weaning. (A,B) Performances in the linear walking test to join the home-cage at PN 51 (no significant differences; n = 11 to 28). (C,D) Histopathology of the cerebellum at PN51 in control and deficient rats, respectively, with the specific marker for Purkinje cells, calbindin-D28 (red), and the nuclear dye DAPI (blue). Any differences were found whatever the layer; molecular layer (*), Purkinje bodies (arrow), granular layer/Purkinje axons (#) (×600 enlargement, bare represent 100 µm); n = 5 per group. (E,F) Evaluation of the learning performances in the aquatic maze at PN 32 to 35. * p < 0.05; ** p < 0.01. n = 8 to 11 per group.

Figure 4

Evaluation of memory retrieval after the switch to normal diet at weaning. (A) Performances in the aquatic maze from PN 80 to PN 330 (escape latency in seconds with pooled results between males and females). (B) Measurement of the CA1 hippocampus layer thickness under a ×200 enlargement of the microscope. (C,D) Histopathology of the CA1 hippocampus layer in control and deficient rats, respectively, with hematoxyline staining (×200 enlargement, bare represent 40 µm). (E,F) Labeling with the specific marker for mature neurons NeuN (red) and the nuclear dye DAPI (blue) (×600 enlargement, bare represent 100 µm). * p < 0.05; ** p < 0.01; n = 8 to 11 per group.

Figure 5

Biochemical analysis of plasma concentration of homocysteine in adults and behavioral evaluation at PN 330. (A) Plasma concentration of Vitamin B12, Vitamin B9, and homocysteine. (B,C) Performances in the linear walking test to join the home-cage at PN 321. * p < 0.05; *** p < 0.001; n = 6 per group.

Figure 6

Evaluation of the learning performances in the water maze at PN 327 to 330 (A,B). (C,D) Histopathology of the CA1 hippocampus layer in control and deficient rats, respectively, with the specific marker for mature neurons NeuN (red) and the nuclear dye DAPI (blue) (×600 enlargement, bare represent 100 µm). * p < 0.05, n = 6 per group.