Long-term exposure to a hypomagnetic field attenuates adult hippocampal neurogenesis and cognition

Adult hippocampal neurogenesis contributes to learning and memory, and is sensitive to a variety of environmental stimuli. Exposure to a hypomagnetic field (HMF) influences the cognitive processes of various animals, from insects to human beings. However, whether HMF exposure affect adult hippocampal neurogenesis and hippocampus-dependent cognitions is still an enigma. Here, we showed that male C57BL/6 J mice exposed to HMF by means of near elimination of the geomagnetic field (GMF) exhibit significant impairments of adult hippocampal neurogenesis and hippocampus-dependent learning, which is strongly correlated with a reduction in the content of reactive oxygen species (ROS). However, these deficits seen in HMF-exposed mice could be rescued either by elevating ROS levels through pharmacological inhibition of ROS removal or by returning them back to GMF. Therefore, our results suggest that GMF plays an important role in adult hippocampal neurogenesis through maintaining appropriate endogenous ROS levels.

(a) Experiments timeline for cell proliferation analysis during HMF or GMF exposure. (b) Representative images of Ki67 + cells in the DG of mice at 0-, 6-, 8-weeks GMF-or HMF-exposure. Scale bar = 200 μm. (c) Quantification of numbers of Ki67 + cells in the DG of GMF-and HMF-exposed mice (GMF, n = 4 mice, HMF, n = 4 mice, GMF versus HMF, two-way ANOVA, F(1, 30) = 22.1, P < 0.0001). All data are presented as mean ± SEM, and error bars were presented in SEM. Data were analyzed by two-way ANOVA, and the two-tailed unpaired t-test for two group comparisons at each time-point. (a) The percentage of BrdU + GFAP + S100βtype 1 aNSCs among total BrdU + cells in the DG of GMF-and HMFexposed mice 1-, 2-, 4-weeks after BrdU injection. (b) The percentage of BrdU + Ki67 + DCXtransient amplifying cells among total BrdU + cells in the DG of GMF-and HMF-exposed mice 1-, 2-, 4-weeks after BrdU injection. (c) The percentage of BrdU + Ki67 + DCX + neuroblasts among total BrdU + cells in the DG of GMF-and HMF-exposed mice 1-, 2-, 4-weeks after BrdU injection. (d) The percentage of BrdU + DCX + NeuN + immature neurons among total BrdU + cells in the DG of GMF-and HMF-exposed mice 1-, 2-, 4-weeks after BrdU injection. (e) The percentage of BrdU + NeuN + mature neurons among total BrdU + cells in the DG of GMF-and HMF-exposed mice 1-, 2-, 4weeks after BrdU injection. (f) The percentage of BrdU + S100β + astrocytes among total BrdU + cells in the DG of GMF-and HMF-exposed mice 1-, 2-, 4-weeks after BrdU injection. GMF, n = 4 mice, HMF, n = 4 mice. All data are presented as mean ± SEM, and error bars were presented in SEM. Data were analyzed by two-way ANOVA, and the two-tailed unpaired t-test for two group comparisons at each time-point. (a) The body weight of adult mice during GMF-or HMF-exposure (GMF, n = 10 mice, HMF, n = 10 mice, GMF versus HMF, two-way ANOVA, F(1, 90) = 1.845, P = 0.1777). (b) Quantification of the relative DG volume of GMF-and HMF-exposed mice (GMF, n = 4 mice, HMF, n = 4 mice, GMF versus HMF, two-way ANOVA, F(1, 23) = 0.4326, P = 0.5172). Data are presented as mean ± SEM, and error bars were presented in SEM. (a) A heatmap of expression levels (log2RPKM; reads per kb of exon per million mapped reads) of differential expression genes in aNSCs isolated from GMF-and HMF-exposed Nestin-GFP mice and their replicates. (b) Volcano plot of differential expression genes. Red and green dots represent probe sets for transcripts expressed at significantly higher or lower levels in aNSCs isolated from GMF-and HMF-exposed mice, respectively. (c) RT-qPCR analysis of mRNA levels of SOD1, SOD2 and SOD3 in GMF-and HMF-cultured aNSCs. GMF, n = 3 mice, HMF, n = 3 mice. (d) Western blot of SOD1, SOD2 and SOD3 in GMF-and HMF-cultured aNSCs. (e) Quantification of western blot analysis on the protein levels of SOD1, and SOD3 in GMF-and HMF-cultured aNSCs. The samples derive from the same experiment (d) and that blots were processed in parallel. GMF, n = 3 mice, HMF, n = 3 mice. (f-h) Elisa analysis for the expression levels of SOD1 (f) , SOD2 (g) and SOD3 (h) protein expression levels in GMF-and HMF-cultured aNSCs. GMF, n = 3 mice, HMF, n = 3 mice. (i-j) The activities of CuZcn-SOD (i) and Mn-SOD (j) in GMF-and HMF-cultured aNSCs. GMF, n = 3 mice, HMF, n = 3 mice. All data are presented as mean ± SEM, and error bars were presented in SEM. P-values were determined from twotailed unpaired t-test; n.s. = not significant. (a) Hydroethidine fluorescence staining in the DG of GMF-and HMF-exposed Nestin-GFP mice with or without DDC treatment. Scale bar = 20 μm. (b) Quantification of hydroethidine fluorescence insensitive of GFP + aNSCs in GFP + cells of GMF-and HMF-exposed Nestin-GFP mice with or without DDC treatment, GMF, n = 410 cells from 4 mice , GMF+DDC, n = 557 cells from 4 mice, HMF, n = 525 cells from 4 mice, HMF+DDC, n = 442 cells from 4 mice. The data were presented in the whisker plot with defined elements, median (center line), upper and lower quartiles (bounds of box), and highest and lowest values (whiskers). P-values were determined from two-tailed unpaired t-test. (c) Representative images of GFP + cells in the DG of GMF-and HMF-exposed Nestin-GFP mice with or without DDC treatment. Scale bar = 200 μm. (d) Quantification of the numbers of GFP + cells in the DG of GMF-and HMF-exposed Nestin-GFP mice with or without DDC treatment, GMF+Vehicle, n = 4 mice, GMF+DDC, n = 3 mice, HMF+Vehicle, n = 4 mice, HMF+DDC, n = 4 mice. Data are presented as mean ± SEM, and error bars were presented in SEM. P-values were determined from two-tailed unpaired t-test; n.s. = not significant.

Supplementary Fig. 8. The effect of DDC treatment on spine development of newborn neurons in GMF-
and HMF-exposed mice.
(a) Representative images of dendritic spines of newborn neurons in the DG in GMF-or HMF-exposed mice with or without DDC treatment. Scale bar = 10 μm. (b) Quantification of the numbers of dendritic of newborn neurons in the DG in GMF-or HMF-exposed mice with or without DDC, GMF+Vehicle, n = 75 from 4 mice, GMF+DDC, n = 74 from 4 mice, HMF+Vehicle, n = 61 from 4 mice, HMF+DDC, n = 102 from 4 mice. The data were presented in the whisker plot with defined elements, median (center line), upper and lower quartiles (bounds of box), and highest and lowest values (whiskers). P-values were determined from two-tailed unpaired t-test. Fig. 9. There were not deficits to open-filed test in HMF-and GMF-exposed mice with or without DDC treatment.
(a-b) There was no difference in the total distance traveled (a) or mean moving speed (b) between GMF-and HMFexposed mice with or without DDC treatment in the open-field task. (c-d) The GMF-and HMF-exposed mice with or without DDC treatment did not exhibit reduced exploration in the margin (c) or center (d) of the arena, shown as the time spent in the margin or center of the arena during the open-field test. GMF+Vehicle, n = 9 mice, GMF+DDC, n = 9 mice, HMF+Vehicle, n = 9 mice, HMF+DDC, n = 8 mice. All data are presented as mean ± SEM, and error bars were presented in SEM. P-values were determined using two-tailed unpaired t-test; n.s. = not significant. a b Supplementary Fig. 10. The promoting effects of return to GMF on ROS levels and GFP + cell population in the DG of HMF-exposed Nestin-GFP mice were fully blocked by APO treatment.
(a) Hydroethidine fluorescence staining in the DG of HMF-exposed Nestin-GFP mice, followed by return to GMF with or without APO treatment. Scale bar = 20 μm. (b) Quantification of hydroethidine fluorescence insensitive of GFP + NPCs in aNSCs of HMF-exposed Nestin-GFP mice, followed by return to GMF with or without APO treatment, GMF, n = 481 cells from 4 mice, HMF, n = 463 cells from 3 mice, HMF+GMF+Vehicle, n = 342 cells from 4 mice, HMF+GMF+APO, n = 368 cells from 4 mice. The data were presented in the whisker plot with defined elements, median (center line), upper and lower quartiles (bounds of box), and highest and lowest values (whiskers). P-values were determined from two-tailed unpaired t-test. (c) Quantification of the numbers of GFP + cells in the DG of HMF-exposed Nestin-GFP mice, followed by return to GMF with or without APO treatment, GMF, n = 4 mice, HMF, n = 3 mice, HMF+GMF+Vehicle, n = 4 mice, HMF+GMF+APO, n = 4 mice. Data are presented as mean ± SEM, and error bars were presented in SEM. P-values were determined from two-tailed unpaired t-test. (a-b) There was no difference in the total distance traveled (a) or mean moving speed (b) during the open-field task in HMF-exposed mice, followed by return to GMF. (c-d) The HMF-exposed mice, followed by return to GMF, did not exhibit reduced exploration in the margin (c) or center (d) of the arena, shown as the time spent in the margin or center of the arena during the open-field test. (e-f) Quantification of the ratio of exploration time on novel location (e) and novel object location (f) in HMF-exposed mice, followed by return to GMF. (g-h) The percentage analysis of freezing behavior during fear conditioning tests (g, contextual test,; h, cue test) in HMF-exposed mice, followed by return to GMF. GMF, n = 8 mice, HMF, n = 10 mice, HMF+GMF, n = 9 mice. All data are presented as mean ± SEM, and error bars were presented in SEM. P-values were determined using two-tailed unpaired t-test. test; n.s. = not significant. a b c d e f g h Supplementary Fig. 12. APO blocked the rescue effects of return to GMF on defective adult hippocampusdependent learning of HMF-exposed mice.
(a-b) There was no difference in the total distance traveled (a) or mean moving speed (b) in HMF-exposed mice, followed by return to GMF with or without APO treatment in the open-field task. (c and d) The GMF-and HMFexposed mice, followed by return to GMF with or without APO treatment, did not exhibit reduced exploration in the margin (c) or center (d) of the arena, shown as the time spent in the margin or center of the arena during the openfield test. (e-f) Quantification of the ratio of exploration time on novel location (e) and novel objective object (f) in HMF-exposed mice, followed by return to GMF with or without APO treatment. (g-h) The percentage analysis of freezing behavior during fear conditioning tests (g, the contextual test; h, cue test) in HMF-exposed mice followed by return to GMF with or without APO treatment. GMF, n = 8 mice, HMF, n = 8 mice, HMF+GMF+Vehicle, n = 8 mice, HMF+GMF+APO, n = 8 mice. All data are presented as mean ± SEM, and error bars were presented in SEM. P-values were determined using two-tailed unpaired t-test. n.s. = not significant. Western blot of SOD1, SOD2 , SOD3 and Actin in GMF-and HMF-cultured aNSCs.