Running in pregnancy transiently increases postnatal hippocampal neurogenesis in the offspring

Abstract

Voluntary wheel running of mice in pregnancy and lactation led to a twofold increase in hippocampal precursor-cell proliferation and in the number of Prox1-expressing lineage-determined cells at postnatal day 8 (P8). At P36, the number of newly generated granule cells approximately doubled, resulting in a 40% higher total number of granule cells in pups from running dams as compared with controls. Cell proliferation at embryonic day 15 (E15), in contrast, was decreased in the progeny of exercising mice, and the birth weight was reduced. At P49, body weight had normalized, and hippocampal neurogenesis was not different between the two groups. mRNA for FGF2 was expressed at higher levels at E15 and P8 in runner pups, whereas VEGF was increased only at E15. Insulin-like growth factor did not show differences at any time point. At P36, no differences for any of the factors were found. Our data indicate that maternal behavior and physical activity affects infantile growth-factor expression and can transiently stimulate postnatal hippocampal development in the offspring.

Fig. 1.

Running in pregnancy has multiple transient effects on the offspring. (A) Average running activity of mice during pregnancy. The average distance (in meters) decreased from 2,500 m/day during early pregnancy to <1,000 m after E10 and very low levels postpartum. (Inset) A pregnant running mouse. (B) Running during pregnancy led to a significant decrease in birth weight of the pups (in grams), measured at P7. In the course of the following weeks, the difference in body weights between CTR and RUN normalized and was absent at P49. (C) At E15, cell proliferation in the hippocampal anlage and the adjacent ventricular and SVZ (see Fig. 2 E and F) was reduced in RUN. (D) Proliferating cells, labeled at P7 with BrdU and examined at P8, increased by 71% in RUN as compared with CTR. (E) Transcription factor Prox1, which is specific to granule cells in the dentate gyrus, was used to further identify lineage-determination in newly generated cells. A significant increase was detected in RUN as compared with CTR. (F) Two different astrocytic markers, glial fibrillary acidic protein (GFAP) and S100β, showed a significant increase in the number of new, BrdU-labeled astrocytes at P8. The greater number of GFAP-positive cells is due to the fact that the putative stem cells of the adult dentate gyrus are GFAP-positive but S100β-negative. (G) At P49, 4 weeks after BrdU injection, the survival of newly generated cells was analyzed. There was a significant increase in BrdU-labeled cells in RUN compared with CTR. (H) Phenotypic analysis (see Fig. 3) of the BrdU-positive cells at P36 revealed an increase by 51% in the number of new neurons (BrdU/NeuN-double-positive) in RUN as compared with CTR. (I) In contrast, the number of newly generated astrocytes at P36 did not reach significance. (J) In animals injected with BrdU at P21, when postnatal hippocampal neurogenesis has largely ceased and the young mice have been weaned from their mother, the same number of surviving BrdU-labeled cells was detectable at P49, suggesting that neurogenesis had returned to control levels in RUN. (K and L) At P49, the number of BrdU/NeuN and BrdU/S100β double-positive cells that had originated from cell proliferation at P21 was not significantly different between RUN and CTR. (M) At P36, the stereologically determined total number of granule cells was significantly greater in RUN than in CTR. (N) The amount of mRNA as a measure of gene expression for three candidate mediators of activity-dependent effects on hippocampal neurogenesis was assessed by quantitative RT-PCR. At P8, FGF2, but not IGF1 and VEGF, was significantly increased in RUN. At E15, FGF2 and VEGF were increased; at P36, no significant differences in growth factor mRNA were detected. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; n/s, not significant

Fig. 2.

Proliferating cells in the dentate gyrus of RUN and CTR pups at P8 and P36. The representative micrographs show an anti-BrdU staining in the granule-cell layer of the dentate gyrus. (Scale bar, 250 μm.) (A and B) At P8, proliferating BrdU-labeled cells were dispersed over the granule-cell layer in RUN (n = 6) and CTR (n = 8). RUN had 71% more BrdU-labeled cells than CTR (P = 0.01). (C and D) At P36, most of the BrdU-labeled cells were detected in the characteristic cell band of the granule-cell layer. Also, at this time point, the total number of granule cells of RUN (n = 5) was 140% of CTR (n = 6), P = 0.001. (E and F) At E15, in RUN, a reduced number of proliferating (i.e., BrdU-labeled) cells in the hippocampal anlage and the adjacent ventricular and SVZ was found. This difference is not obvious in the photomicrograph but was statistically significant upon quantification (see Fig. 1C). (G and H) At P8, no obvious difference (as in the hippocampus, see A and B) in the number of BrdU-labeled cells was found in the SVZ. However, the experiment was not designed to study the SVZ quantitatively, and no strong conclusions should be drawn from the impression gained from these images.

Fig. 3.

Confocal microscopic and DNA stain bisbenzemide analysis of granule cells and astrocytes in the dentate gyrus (DG) of pups from running mothers (RUN). Within each panel, the antigens investigated are listed in the order red, blue, and green. (A) Distribution of granule cells in the dentate gyrus at P8 was identified by their expression of the neuronal transcription factor Prox1. Newly generated granule cells were identified by immunoreactivity for proliferation marker BrdU (red) and Prox1 (blue). One day after BrdU application, the number of Prox1-expressing newly generated granule cells increased significantly in RUN compared with CTR. (B and C) At P36, survival of newly generated cells was quantified by their incorporation of BrdU (red). Neurons and astrocytes were characterized by their expression of NeuN (blue) or S100β (green). The survival rate of new neurons and astrocytes was significantly increased in RUN compared with controls. (D) Total granule-cell numbers were determined by using stereology (optical fractionator) based on DNA stain bisbenzemide (Hoechst 33258; see inset for higher magnification). Increased neurogenesis in the DG in RUN resulted in an absolute increase in the total number of granule cells at P36. [Scale bar in D for all panels; 100 μm (A and B), 30 μm (C), and 200 μm (D).]