Research report
Developmental vitamin D deficiency alters learning in C57Bl/6J mice

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Abstract

Epidemiological studies have highlighted a season of birth effect in multiple sclerosis and schizophrenia. As a result, low prenatal vitamin D has been proposed as a candidate risk factor for these brain diseases, with cognitive impairments. In order to further investigate the long-term consequences of a transient gestational hypovitaminosis D, we used a mouse developmental vitamin D (DVD) deficiency model. Female C57Bl/6J mice were fed a vitamin D-free diet for 6 weeks prior to conception and during gestation. At birth, dams and their offspring were fed a normal vitamin D-containing diet. The adult offspring underwent a learning test based on olfactory cues, at 30 weeks and 60 weeks of age. In addition, using magnetic resonance imaging (MRI), volumes of cerebrum, hippocampus and lateral ventricles were measured at 30 weeks and 70 weeks of age. We found that DVD-deficient mice, when compared to control animals at Week 30, displayed impaired learning and smaller lateral ventricles. At Weeks 60–70, both groups deteriorated when compared to young mice and no significant difference was observed between groups. This study confirms that transient prenatal vitamin D deficiency alters brain development and functioning and induces cognitive impairments in the young adult offspring.

Introduction

It is now well established that vitamin D, a seco-steroid hormone which is produced in the animal epidermis under UVB radiation, exerts patent effects on neural cells [14]. Furthermore, an impaired supply of vitamin D is a risk factor for various brain diseases [18]. A high prevalence of vitamin D deficiency has been reported in infants, children, and adolescents from various countries (for a review [26]) and it has been suggested that up to 1 billion people around the world may have vitamin D deficiency or insufficiency [24].

Among the general population, pregnant women are at risk of hypovitaminosis D. A reduced outdoor activity, associated with increased needs of the foetus, may lead to a diminished supply of vitamin D [23], [36]. A large US study reported that, of the women aged 20–39 (peak ages for child-bearing), 12% had low serum 25-hydroxyvitamin D levels [34]. Hypovitaminosis D can even reach endemic proportions in specific communities: vitamin D deficiency was observed in 82.5% of women wearing concealing clothes [6].

The Barker hypothesis postulates that some adult diseases may originate from aberrant environmental imprints during development [4]. This theory has been substantiated by numerous studies (for a review [41]). In regard to schizophrenia, an increased risk of developing this disease is observed in individuals born in winter or spring [40], [48] while low maternal vitamin D levels are associated with a higher risk of schizophrenia in offspring [38]. Similarly, it has been demonstrated that the risk of multiple sclerosis is greater for people born in May and lower for those born in November [17], [53].

Using a developmental vitamin D (DVD) deficiency model, we showed that a transient prenatal hypovitaminosis D adversely affects rat brain development. Enlarged lateral ventricles, decreased expression of NGF, GDNF and p75NTR (low affinity neurotrophin receptor) and signs of delayed differentiation were observed in DVD-deficient neonates [13]. The decrease in NGF persisted in the adult offspring along with enlarged lateral ventricles and increased cell packing in pre-frontal cortical areas [19]. We also demonstrated a robust and consistent downregulation of transcripts and proteins involved in cytoskeleton maintenance, molecular transport of organelles and synaptic plasticity [1], [12].

These molecular abnormalities were associated with altered behaviours. DVD-deficient adult rats and mice exhibited hyperlocomotion in a novel environment [7]. However, while rats displayed decreased exploratory behaviour and increased rearing [8], [9], [15], [30], mice presented an increased exploration and no modified rearing [22]. In addition, DVD-deficient rats showed signs of (i) an impaired latent inhibition, a process by which pre-exposure to a to-be-conditioned stimulus retards the subsequent learning of a conditioned association to that stimulus [5] and (ii) changes of synaptic plasticity in the dentate gyrus [21].

In line with abnormalities induced by developmental vitamin D deficiency, cognitive defects have also been reported when vitamin D receptor (VDR) is ablated. The VDR, expressed in the limbic system [49] and more generally in the whole nervous system [16], [45], is a ligand-regulated transcription factor that recognizes cognate vitamin D response elements in more than 900 genes, including genes involved in cognitive functions [50]. When the VDR is ablated, mice display high anxiety [29] and neophobia [42], in addition to motor deficits [28]. When the VDR function is disturbed by single nucleotide polymorphisms, elder patients exhibit changes in cognitive functions and depressive symptoms [33]. In addition, several clinical studies have suggested an association between 25-hydroxyvitamin D levels and cognitive performance (for a recent review [2]).

The first aim of this study was to assess learning in the DVD-deficient C57Bl/6J mice, in young and in aged adult. We used a hippocampus-dependent test based on olfactory cues, namely the olfactory tubing maze [47]. Then, with the help of Magnetic Resonance Imaging (MRI), we measured hippocampus, lateral ventricle and cerebrum volumes in order to evaluate the maternal vitamin D deficiency impact on gross morphology and a possible correlation with the observed behaviour.

Section snippets

Developmental vitamin D deficiency model

All procedures were performed according to the French law on Animal Care Guidelines and the Animal Care Committee of University Aix-Marseille II approved our protocols. All animals were weighted before each experiment.

Female C57Bl/6J mice (Charles River, France) were housed in a single holding room, at a constant temperature of 21 ± 2°C and 60% relative humidity, on a 12 h light–dark cycle. Food and water were provided ad libitum. Developmental vitamin D deficiency was achieved by (i) feeding

Learning assessment

Comparisons between 30-week-old DVD-deficient mice and control mice did not show any overall impairment of performance across the five training sessions (MANOVA; F(1,16) = 1.03, ns). However, there was an effect of maternal diet on the fifth day, with a significant deficit for the DVD-deficient mice (ANOVA: F(1,16) = 5.08, p < 0.05) (Fig. 1A). DVD-deficient adult mice reached a mean score of 70% ± 2.69 (±S.E.M.) while control mice achieved a mean score of 80% ± 3.66. During the five training sessions,

Discussion

Maternal vitamin D deficiency resulted in anatomical and behavioural changes. Two findings arise from this study: at Week 30: (i) impaired learning during an associative hippocampal-dependent memory task and (ii) smaller lateral ventricles were observed in DVD-deficient C57Bl/6J mice, when compared to control mice. However, at Week 60 and Week 70, no significant differences were observed between both groups.

Given that (i) rodents rely mostly on olfaction for their daily behaviours and (ii) mice

Acknowledgements

We gratefully acknowledge Alarme, ARSEP, Demain Debout Foundations, Fondation de l’Avenir and IRME (Institut pour la Recherche sur la Moelle épinière et l’Encéphale) for their financial support.

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