Vitamin D is crucial for maternal care and offspring social behaviour in rats

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   Figure 1

   Vitamin D levels altered maternal care parameters in the first postnatal week. (A) Maternal vitamin D deficiency had a significant effect on the amount of time the dam spent licking and grooming her pups, but there was no effect of day or interaction between diet and day. (B) Combining all the data points from PND3 to 8 demonstrates the extent to which vitamin D deficiency decreased total licking and grooming from PND3 to 8. (C) At PND7 there were no differences in the latency for control or vitamin D-deficient dams to retrieve their pups during the pup retrieval test. Bars indicate mean + s.e.m. (n = 12 control dams and 10 vitamin D-deficient dams). Significant differences; two-way ANOVA or t-test, *P < 0.05, **P < 0.01.
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   Figure 2

   Ultrasonic vocalisations (USV) of PND12 pups upon 5-min repeated maternal separation. (A) Ultrasonic vocalisation total call number was markedly increased in vitamin D-deficient pups for the first (Sep 1), but not the second (Sep 2) maternal separation period. For each separation period, the calls were also classified based upon the peak frequency of the call (f), shown in panels (B) and (C). However, these did not differ. Bars indicate mean + s.e.m. (n = 8 for both control and vitamin D-deficient groups). Significant differences: Kurskal–Wallis, **P < 0.01.
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   Figure 3

   FOXP2, but not TH, immunolabelling was decreased in vitamin D-deficient pups in comparison to control at PND12. FOXP2 immunolabelling and analysis is shown in panels (A, B, C and D). (A) Coronal schematic representation of region of interest for immunohistochemical labelling of FOXP2 in the cortex of PND12 animals. The box represents the region shown in example (B) control and (C) vitamin D-deficient images of FOXP2 immunolabelling in the cortex and was used for (D) semi-quantitative immunointensity analysis. Tyrosine hydroxylase (TH) immunolabelling and analysis is shown in panels (E, F and G). Example substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) immunolabelling in (E) control and (F) vitamin D-deficient animals. (G) Semi-quantitative analysis revealed no differences in TH immunointensity. Bars show mean + s.e.m., n = 6–8 per diet group. Significant differences: t-test, *P < 0.05.
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   Figure 4

   Adult social and cognitive behaviours were decreased animals exposed to early life vitamin D deficiency in comparison to control. Social interaction test: (A) example exploration traces percentage of time spent in the social cue chamber containing the novel rat for (i) vitamin D-deficient offspring and (ii) control offspring. Social preference was quantified by (B) percentage of time socially interacting in the social cue chamber. Cognitive outcomes: (C) novel object recognition discrimination ratio after 1 h separation, and (D) object-in-place discrimination ratio. Sucrose preference test assessment of anhedonia revealed no differences (E) as assessed by preference for sucrose solution over water. Open field test outcomes were unaltered between the groups during standard testing days (days 1–3) and following restraint stress (days 4 and 5), (F) total distance travelled and G) centre/periphery exploration time ratio. While exploration of the elevated plus maze could not be assessed, grooming behaviour was scored. Early life vitamin D deficiency increased total grooming duration (H) but not grooming frequency (I). Bars represent mean + s.e.m., n = 10–15 per diet group. Significant differences: t-test or Kruskal–Wallis, *P < 0.05.
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   Figure 5

   MRI volumetric analysis of brains from ~PNW16 control and vitamin D-deficient animals, and relative changes in gene expression. Example images illustrating the marked differences in lateral ventricle volumes of (A) control and (B) vitamin D-deficient animals at a comparable section level of the brain. (C) Volume of brain regions of the cerebellum (Cer), hippocampus (Hipp), corpus callosum (CC) and lateral ventricles (LV) normalised to total brain volume. Early life vitamin D deficiency caused a downregulation in some genes involved in dopaminergic pathways. Relative gene expression to house-keeping genes for whole brain on postnatal day 1 (PND1) for: (D) GABA synthesis enzyme, Gad1; (E) dopamine receptor type 2, Drd2. Adult (4 months old) brain regional gene expression for (F) dopamine receptor type 2, Drd2, in the cerebrellum; and (G) NMDA receptor subunit, Grin2b, in the mesencephalon. Bars show mean + s.e.m., n = 8 for both control and vitamin D-deficient groups. Significant differences: t-test, *P < 0.05, **P < 0.01.
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   Figure 6

   Early life vitamin D deficiency altered some genes involved in glucocorticoid pathways. In postnatal day 1 (PND1) animals, vitamin D deficiency caused a reduction in (A) Hsd11b2 and (B) Nr3c2 levels in the whole brain. There was an (C) increase in Tsc22d3 expression in vitamin D-deficient animals, and (D) no changes in serum corticosterone levels, at PND1, weaning, and in adulthood (4 months). Bars show mean + s.e.m., n = 8 for both control and vitamin D-deficient groups. Significant differences: t-test, *P < 0.05.

• © 2018 Society for Endocrinology