Maternal Vitamin D Prevents Abnormal Dopaminergic Development and Function in a Mouse Model of Prenatal Immune Activation

Abstract

Dysfunction in dopamine (DA) systems is a prominent feature in schizophrenia patients and may result from the abnormal development of mesencephalic (mes)DA systems. Maternal immune activation (MIA) and developmental vitamin D (DVD)-deficiency both induce schizophrenia-relevant dopaminergic abnormalities in adult offspring. In this study, we investigated whether maternal administration of the vitamin D hormone (1,25OHD, VIT_D) could prevent MIA-induced abnormalities in DA-related behaviors and mesDA development. We administrated the viral mimetic polyriboinosinic-polyribocytidylic (poly (I:C)) simultaneously with 1,25OHD and/or their vehicles, to pregnant mouse dams at gestational day 9. Maternal treatment with VIT_D prevented MIA-induced hypersensitivity to acute DA stimulation induced by amphetamine, whereas it failed to block prepulse inhibition deficiency in MIA-exposed offspring. MIA and VIT_D both reduced fetal mesDA progenitor (Lmx1a + Sox2+) cells, while VIT_D treatment increased the number of mature (Nurr1 + TH+) mesDA neurons. Single-cell quantification of protein expression showed that VIT_D treatment increased the expression of Lmx1a, Nurr1 and TH in individual mesDA cells and restored normal mesDA positioning. Our data demonstrate that VIT_D prevents abnormal dopaminergic phenotypes in MIA offspring possibly via its early neuroprotective actions on fetal mesDA neurons. Maternal supplementation with the dietary form of vitamin D, cholecalciferol may become a valuable strategy for the prevention of MIA-induced neurodevelopmental abnormalities.

Figure 1

The effects of single and combined MIA and VIT_D on prepulse inhibition and amphetamine sensitivity in adult offspring. (A) Prepulse inhibition (PPI) of the acoustic startle reflex was used to investigate sensorimotor gating in adult offspring. %PPI is shown for three startling amplitudes (100, 110 and 120 dB_A, which are noted as P-100, P-110 and P-120, respectively) and prepulse intensities (71, 77 and 83 dB_A, corresponding to +6, +12 and +18 dB_A above background white noise). Poly(I:C) exposure impaired P-110dB_A PPI. The bar plots represent the mean %PPI for all prepulse and pulse levels. All values were medians ±SEM. ^*p < 0.05, reflecting the significant main effect of MIA on mean % PPI; ^***p < 0.001, reflecting the significant main effect of MIA in the P-110 condition. (B) The line plot shows total distance travelled in an open field per bin (=5 min) during initial habituation and subsequent saline administration periods, followed by the amphetamine (AMPH; 2.5 mg/kg, i.p.) administration phase. ^*p < 0.05, reflecting the significant difference between POL-VEH and CON-VEH offspring (AMPH bins 1–9); ⁺p < 0.05, reflecting the significant difference between POL-VEH and POL-Vit_D offspring (AMPH bins 11 and 12). All values were means ± SEM.

Figure 2

The effects of single and combined MIA and VIT_D on early mesencephalic dopamine (mesDA) cell number. (A–D) Fate mapping of developing mesDA cells in a medial coronal mesencephalic (MES) sections at gestational day (GD) 11. Markers are the nuclear dye DAPI (blue, A), mesDA specification factor Lmx1a (green, B), neuronal progenitor maker Sox2 (red, C), and a channel-merged image (D). (A’–D’) Higher magnification images of the dashed outlined boxes representing the floor plate (FP) from (A–D) accordingly. Lmx1a specifically marked mesDA cells in the FP dorsoventrally from ventricular zone (VZ), intermediate zone (IZ) to mantle zone (MZ). MesDA progenitors (Lmx1a+Sox2+) were primarily located in the proliferative VZ, in contrast, post-mitotic (Lmx1a+Sox2−) mesDAs were mostly located in IZ and MZ. (E) The cell number of these two mesDA subpopulations were counted using CellProfiler software at 60 µm intervals along the anterior-posterior (A–P) axis. The numbers of mesDA progenitors (Lmx1a+Sox2+) were reduced by all treatments relative to the control (CON-VEH) (p’s < 0.05). (F) Post-mitotic (Lmx1a+Sox2−) mesDA number was not altered by any treatment. (G–J) Fate mapping of post-mitotic mesDA subgroups that are characterized by Nurr1 and TH in medial coronal MES sections at GD11. Markers are the nuclear dye DAPI (blue, G), Nurr1 (magenta, H), TH (green, I), and a channel-merged image (J). (G’–J’) Higher magnification images of the dashed outlined boxes representing FP from (G–J) accordingly. Immature Nurr1+ mesDAs were primarily located in the IZ and MZ. Mature (Nurr1+ TH+) mesDAs were primarily located in MZ. (K) There were no significant differences of immature mesDA number among treatment groups (p’s > 0.05). (L) VIT_D treatment itself increased mature (Nurr1+ TH+) mesDA number compared to its vehicle (VEH) (p < 0.05). VIT_D treatment itself (CON-VIT_D) particularly increased mature mesDA number in the posterior MES compared to control (CON-VEH) (p’s < 0.05). All values were means ± SEM. ^*P < 0.05. n.s., represents not statistically significant. Scale bars: 100 µm (A–D and G–J); 50 µm (A’–D’ and G’–J’).

Figure 3

The effects of single and combined MIA and VIT_D treatments on the coronal positioning of mature mesencephalic dopamine neurons (mesDAs). (A) Schematic image of registered floor plate (FP) in a medial coronal mesencephalic (MES) section with mediolateral (ML, x) and dorsoventral (DV, y) positions of cells. The coordinates (x₀, y₀) were chosen as the most ventral point of ventricle (V) along the midline (*). The black dot represents the mean center positioning (x_n, y_n) of a representative mesDA nucleus (ellipse). The ML positioning (x) of mesDA cells was measured bilaterally as the absolute distance from the center of mesDA nucleus (x_n) to the coordinate (x₀) = ABS (x_n−x₀). The DV positioning (y) of mesDA cell was calculated as the distance from the center of mesDA nucleus (y_n) to the coordinate (y₀) = (y₀−y_n). (B) An example of mesDA positioning in the FP. Yellow dots represent mesDA progenitors (Lmx1a+Sox2+), and green dots represent post-mitotic (Lmx1a+Sox2−) mesDAs. (C) A representative FP section showing cells that were triple-labeled by DAPI (blue), Lmx1a (green) and Sox2 (red). No significant effects of MIA or VIT_D treatment were noticeable for the mean ML (D) or DV (E) positioning of mesDA progenitors (p’s > 0.05). (F) There were no significant effects of MIA or VIT_D treatments on the mean ML positioning of post-mitotic (Lmx1a+Sox2−) mesDAs (p’s > 0.05). (G) There were significant interactions between MIA or VIT_D treatments on the average DV positioning of post-mitotic (Lmx1a+Sox2−) (p < 0.05). MIA treatment (POL-VEH) reduced the DV positioning of post-mitotic mesDAs compared to its control (CON-VEH) (p < 0.05). (H) A representative FP of a coronal MES section showing cells that were triple-labeled by DAPI (blue), Nurr1 (magenta) and TH (green). There were no significant effects of MIA or VIT_D treatment for the mean ML (I) or DV (J) positioning of immature (Nurr1+TH−) mesDAs in the FP (p’s > 0.05). (K) MIA treatment (POL) reduced the mean ML positioning of mesDAs relative to CON (p < 0.05). (L) There were significant interactions between MIA x VIT_D treatments in the average DV positioning of mature (Nurr1+TH+) mesDAs (p < 0.05). Post hoc comparison revealed MIA treatment (POL-VEH) decreased the mean DV positioning relative to the control (CON-VEH) (p < 0.05). Additionally, the co-treatment of VIT_D restored the mean DV positioning of mature mesDAs in POL-VIT_D group compared to POL-VEH group (p < 0.05). All values were means ± SEM. ^*p < 0.05. n.s. represents not statistically significant. Scale bars: 50 µm.

Figure 4

The effects of single and combined MIA and VIT_D treatment on the expression of factors important in the development of mesDA neurons. (A) A representative floor plate (FP) of coronal MES section showing mesDA cells expressing Sox2 (red). (B) Neither MIA or VIT_D treatment altered the mean intensity of Sox2 in mesDA progenitors (Lmx1a+Sox2+) (p’s > 0.05). (C) Bar plots represent the average fluorescence of Sox2 in mesDA progenitors among treatment groups. Distribution analysis representing a nonlinear regression to predict the Gaussian distribution of mesDA progenitor number relative to the intensity of Sox2 expression. (D) A representative FP of coronal MES section showing post-mitotic mesDA cells expressing Lmx1a (green). (E) VIT_D treatment significantly increased the mean intensity of Lmx1a in mesDAs compared with other treatment groups (p’s < 0.05). (F) Bar plots represent the average expression of Lmx1a among treatment groups. Distribution analysis showed VIT_D treatment itself increased the number of cells expressing higher levels of Lmx1a compared to other treatment groups (p’s < 0.05). (G) A representative FP of coronal MES section showing post-mitotic mesDA cells expressing Nurr1 (magenta). (H) VIT_D treatment significantly increased the expression of Nurr1 in post-mitotic (Nurr1+) mesDAs (p < 0.05). (I) Bar plots represent the average fluorescence of Nurr1 among treatment groups. Distribution analysis showed VIT_D treatment increased the number of cells expressing higher levels of Nurr1 compared to VEH (p < 0.05). (J) A representative FP of coronal MES section showing mature mesDAs expressing TH (green). (K) VIT_D treatment increased the average fluorescence of TH in mature (Nurr1+TH+) mesDAs. (L) Bar plots represent the average TH mean intensity in mature mesDAs among treatment groups. The distribution analysis verified that VIT_D treatment increased the number of mature (Nurr1+TH+) mesDAs that expressed higher levels of TH compared to VEH (p < 0.01). All values were means ± SEM. ^*p < 0.05, ^**p < 0.01. Scale bars: 50 µm.