Behavioural Effects of Adult Vitamin D Deficiency in BALB/c Mice Are not Associated with Proliferation or Survival of Neurons in the Adult Hippocampus

Abstract

Epidemiological studies have shown that up to one third of adults have insufficient levels of vitamin D and there is an association between low vitamin D concentrations and adverse brain outcomes, such as depression. Vitamin D has been shown to be involved in processes associated with neurogenesis during development. Therefore, the aim of this study was to test the hypothesis that adult vitamin D (AVD) deficiency in BALB/c mice was associated with (a) adult hippocampal neurogenesis at baseline, b) following 6 weeks of voluntary wheel running and (c) a depressive-like phenotype on the forced swim test (FST), which may be linked to alterations in hippocampal neurogenesis. We assessed proliferation and survival of adult born hippocampal neurons by counting the number of cells positive for Ki67 and doublecortin (DCX), and incorporation of 5-Bromo-2'-Deoxyuridine (BrdU) within newly born mature neurons using immunohistochemistry. There were no significant effects of diet on number of Ki67+, DCX+ or BrdU+ cells in the dentate gyrus. All mice showed significantly increased number of Ki67+ cells and BrdU incorporation, and decreased immobility time in the FST, after voluntary wheel running. A significant correlation was found in control mice between immobility time in the FST and level of hippocampal neurogenesis, however, no such correlation was found for AVD-deficient mice. We conclude that AVD deficiency was not associated with impaired proliferation or survival of adult born neurons in BALB/c mice and that the impact on rodent behaviour may not be due to altered neurogenesis per se, but to altered function of new hippocampal neurons or processes independent of adult neurogenesis.

Fig 1. Representative images of BrdU incorporation within mature neurons in the dentate gyrus.

A representative image of the dentate gyrus from a Non-Runner (a) and a Runner (b). A close up image from the section of the dentate gyrus from the Non-Runner (c), and the Runner (g), DAPI stain showing nuclei (d and h), NeuN stain showing mature neurons (e and i) and BrdU staining showing newly born cells (f and j). The line arrow points to a mature neuron (stained with NeuN) with complete BrdU incorporation, these cells have not undergone cell division after the uptake of BrdU. The arrow head points to a mature neuron (stained with NeuN) with only partial BrdU incorporation, these cells have divided following BrdU injection or were born late in the accumulation window. The star points to a cell with BrdU incorporation but no co-localisation with NeuN and is therefore not a neuron (not analysed). Blue–DAPI, Green–NeuN, Red–BrdU. A representative image of Ki67 staining (arrow, k and l) and control (m and n). Blue–DAPI, Red–Ki67.

Fig 2. Results for the activity monitor, forced swim test (FST), and Ki67⁺, BrdU⁺ NeuN⁺, and DCX⁺ cell counts.

The distance travelled in the activity monitors is shown in (a) (n = 13–15 per group). There was no significant effect of Diet but there was a significant main effect of Running. Runners moved less than Non-runners. Immobility time during the FST is shown in (b) (n = 7–8 per group). There was a main effect of both Diet and Running. Wheel running significantly reduced immobility time; moreover AVD deficiency also reduced immobility time. The number of Ki67⁺ is shown expressed as a percent of control non-runner values (c) (n = 8 per group). There was no significant effect of diet but there was a significant effect of Running. Runners had higher numbers of Ki67⁺ cells compared to Non-runners. There was no significant difference between control and AVD-deficient mice. BrdU⁺ NeuN⁺ cell counts expressed as a percent of control non-runner values is shown in (d) (n = 8 per group). There was a significant increase in the number of BrdU⁺ NeuN⁺ cells following voluntary running. However, there was no significant effect of Diet on baseline cell numbers or cell numbers stimulated by voluntary running. The number of DCX expressed as a percent of control non-runner values is shown in (e) (n = 7 per group). Where main effect is significant, p-value is given on graph. Mean ± SEM (* p < 0.05).