Vitamin D: Brain and Behaviour

Darryl Eyles *1,2

1. Queensland Centre for Mental Health Research, The Park Centre for Mental

Health, Wacol, Q4076, Australia.

2. Queensland Brain Institute, University of Queensland, St Lucia, Q4076

Australia.

* Corresponding Author:

Prof Darryl Eyles

Neurobiology Laboratory Director

Queensland Brain Institute, University of Queensland, Brisbane QLD 4072 Australia

Queensland Centre for Mental Health Research, The Park Centre for Mental Health
Wacol QLD 4076 Australia

phone (QBI) +61 7 3346 6370

fax +61 7 33466301

www.qcmhr.uq.edu.au

www.qbi.uq.edu.au

email address: - [email protected]

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jbm4.10419

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Summary

It has been 20 years since we 1st proposed vitamin D as a “possible” neurosteroid.(1)

Our work over the last 2 decades, particularly results from our cellular and animal

models has confirmed the numerous ways in which vitamin D differentiates the

developing brain. As a result vitamin D can now confidently take its place amongst all

other steroids known to regulate brain development.(2) Others have concentrated on the

possible neuroprotective functions of vitamin D in adult brains. Here these data are

integrated, and possible mechanisms outlined for the various roles vitamin D appears to

play in both developing and mature brains and how such actions shape behavior. There

is now also good evidence linking gestational and/or neonatal vitamin D deficiency with

an increased risk of neurodevelopmental disorders such as schizophrenia and autism

and adult vitamin D deficiency with certain degenerative conditions. Indeed the author of

this review has been heavily invested in this analytical epidemiology. However in this

mini-review I will not be discussing this clinical epidemiology but rather focusing on what

we have learned over this time period regarding the genomic and non-genomic actions

of vitamin D in shaping brain development, neurophysiology and behaviour in animal

models.

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Key Words

Brain, Development, Vitamin D deficiency, Neuroprotection

I. Introduction
II. Vitamin D signaling and metabolism in the brain
III. Developmental vitamin D (DVD)-deficiency and brain development
IV. Developmental vitamin D (DVD)-deficiency and animal behavior
V. Adult vitamin D (AVD)-deficiency/supplementation and brain and behavior
VI. Vitamin D regulates essential processes in normal brain development and
function and is neuroprotective in adult brains
VII. Conclusions

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I. Introduction

Readers of this, and other contributions to this issue will be aware of the wide range of

non-skeletal targets for vitamin D. In particular the last 20 years have been a fertile

period for the investigation of vitamin D and its diverse functions in the brain. The

distribution of the vitamin D receptor (VDR) and the enzyme associated with the

synthesis of the active form of the hormone 1-alpha hydroxylase (CYP27B1) have been

mapped in human brain,(3) along with studies showing the VDR is present in numerous

brain cells such as oligodendrocytes, astrocytes, microglia and neurons.(2,4,5)

Experimentally-induced variations in vitamin D status have been shown to affect brain

cell differentiation, neurotrophin expression, cytokine regulation, neurotransmitter

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synthesis, intracellular calcium signaling, anti-oxidant activity, and the expression of

genes/proteins involved in neuronal structure, physiological function and metabolism.(6,7)

Therefore, perhaps it comes as no surprise that vitamin D status should be related to a

number of clinical brain disorders. For more than a decade now inadequate levels of

vitamin D have been linked with numerous adverse brain related outcomes. In particular

developmental vitamin D (DVD) deficiency has been linked with schizophrenia (8,9) and

more recently autism.(10-12) Adult vitamin D (AVD) deficiency has also been linked with

schizophrenia, Alzheimer’s disease, dementias and adult disorders of cognition (for

review see (13)). There are also strong links with Parkinson’s disease.(14)

It is not the purpose of this mini-review to concentrate on this clinical epidemiological

literature. For detailed reviews of these associations I refer the reader to two excellent

recent summaries.(13,15) I will return to a discussion of the interpretation of this clinical

literature at the end of article. Rather my purpose here is to focus on the latest

preclinical literature modelling these epidemiological links and discuss plausible

biological mechanisms. I will present convincing evidence connecting vitamin D

deficiency in animals with behavioural phenotypes of relevance to the aforementioned

clinical conditions. The biological plausibility that low levels of vitamin D adversely affect

brain development and function is now well-established. Our task now, is to discover

exactly how low levels of vitamin D change the function of specific brain cells/ circuits

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predisposing an individual to develop such disorders and to see if correcting vitamin D

status can ameliorate phenotype/symptom severity.

Like many of the other contributors to this special edition I would like to honour Tony

Normans legacy. When I went to my 1st Vitamin D workshop in Maastricht in 2003 I felt

like a total imposter. What was a neuroscientist doing in an endocrine meeting where to

the best of my knowledge no one had even mentioned the brain before? I acknowledge

Tony for creating and sustaining this meeting which for me at least has become a truly

collegial environment for collaboration and for allowing us a continuing platform to

communicate our research.

II. Vitamin D signaling and metabolism in the brain

The major circulatory form of vitamin D, 25-hydroxyvitamin D3, (25(OH)D3) and its active

hormonal form, 1,25-hydroxyvitamin D3, 1,25(OH)2D3 are present in the brain.(16,17)

Whilst the exact concentrations are debatable, they are likely to be much lower than

those levels found in blood. Various technical issues in their extraction and method of

quantification make claims of absolute amounts difficult at this time. We have dealt

extensively with these issues elsewhere.(15)

Immunohistochemical evidence for the VDR is far stronger. The VDR has been

confirmed in human, mouse, rat, chick and zebrafish brains.(3,18-22) VDR’s in brain are

also functional, specifically binding to DNA response elements when liganded.(23) In

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response to claims from some authors that immunological detection of the VDR was

open to errors,(24,25) we have provided unambiguous evidence via mass-

spectrophotometric protein sequencing of electrophoretically resolved proteins from

adult rodent brains which identified five unique VDR peptides with a confidence interval

greater than 99%.(26) The regional organization of VDR in human brain is remarkably

consistent with that published for the rat. For example, VDR expression in both rat and

human cerebellum is restricted to the granule cells and was completely absent from

Purkinje cells.(3,19) In the human hippocampus, VDR immunoreactivity was strongest in

CA1 and CA2 pyramidal cells with a marked reduction within CA3,(3) a finding replicated

in rats by two separate groups.(19,21) Within both the rat and human hypothalamus the

most densely labeled nuclei were the supraoptic and paraventricular nuclei.(3,18) VDR

immunoreactivity was also completely absent in the large, presumably cholinergic

neurons from the nucleus basalis in both rat and human brains.(27) Finally the

concentration of VDR protein within the large dopaminergic neurons of the substantia

nigra has now been confirmed in both rat and human brain.(28) Most importantly VDR in

brain is assumed to be functional in that it is able to specifically bind DNA response

elements when bound to ligand.(23) This close cross-species overlap in VDR distribution

validates the use of rodents in modelling vitamin D related brain outcomes.

The temporal nature of VDR expression in the developing brain has been qualitatively

mapped in both rat(29) and mouse brain.(30) The immunohistochemical presence of VDR

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emerges on embryonic day (E)12 in rats and E11.5 in mouse. As found in adult brain

there is a broad distribution across a variety of brain regions. In the developing rat brain,

the VDR appeared to be localized in differentiating fields.(29) This may be highly relevant

given the role of vitamin D as a potent differentiation agent in a variety of cell types.(31)

We have provided more evidence that VDR signaling may be relevant to brain cell

proliferation by identifying intense VDR immunohistochemical presence in the

ependymal surface of the lateral ventricles in neonatal rats.(32) This is a site that

represents the richest source of cell division in the postnatal brain.

Our initial studies of the time course for VDR protein and mRNA expression in the rat

brain showed a general increase between embryonic days 15 and 23.(33) Since then we

have studied the ontogeny of the VDR in developing rodent brain at the

immunohistochemical, mRNA and protein levels. We confirmed earlier findings that the

VDR is present at the early embryonic age of E12, but this staining did not appear to be

cellular. By E15, clear punctate staining was obvious in the nucleus of dopaminergic

neurons gaining a mature pan-nuclear appearance by birth in these cells.(28) This

pattern of expression was largely confirmed at the mRNA and protein levels.(28) We

have confirmed VDR protein and mRNA were present in the neonatal brain in a

subsequent study.(26) The appearance of the VDR and its increasing expression in the

embryo coincides with the onset of neuronal differentiation in the developing brain.

While a causal association cannot be directly established from these anatomical

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studies, they are consistent with vitamin D operating via its receptor to either directly or

indirectly mediate features of neuronal apoptosis and cell cycle.

Finally the enzyme which catalyzes conversion of 25(OH)D3 to the active or hormonal

form of vitamin D, 1,25(OH)2D3, CYP27B1, is clearly present in the fetal human (34) and

adult brain(3,35) suggesting local production of the active hormone may be possible in

human brain. The enzyme was detected in the cytoplasm of both neurons and non-

neuronal cells.(3) As with the VDR, the pattern of expression of this enzyme had regional

and sub-regional specificity. The regions that stained the strongest were the supraoptic

and paraventricular nuclei within the hypothalamus and the substantia nigra.

Hydroxylation of 25(OH)D3 at position 24 by the enzyme CYP24A1 is a major catabolic

pathway for vitamin D metabolites. There appears to be a selective distribution of

CYP24A1 and CYP27B1 in non-neuronal cells with CYP24A1 primarily found in

astrocytes and CYP27B1 in microglia within brain.(36)

III. Developmental vitamin D (DVD)-deficiency and brain development

The effects of manipulating vitamin D signaling on brain development have been

measured by either genetically ablating the VDR or CYP27B1 or in models of dietary

deficiency. Here we will not discuss brain functional data from the genetic models

further as the physiology of these animals is often compromised thus distorting behavior

(see section IV below). However there is abundant research from models of dietary

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restriction. Our group was the first to create a dietary model of Developmental Vitamin D

(DVD) deficiency in rodents specifically to examine developmental brain related

outcomes.(37-39) These vitamin D deficient dams and DVD-deficient offspring have

normal calcium and phosphate levels i.e. neither the dams nor their offspring have a

rickets-like phenotype.(40) Since we developed this model in rats in 2003 it has been

adapted in both other rat or mouse strains to examine the long-term neuropsychiatric

outcomes of DVD-deficiency.(41-44) In summarising findings from such models they

would all appear to produce changes in brain cell differentiation, anatomy,

neurotransmitter production and gene and protein expression

DVD-deficient rat embryos have increased brain cell proliferation,(32,38,45) reduced

apoptosis(38,44,45) along with corresponding changes in cell-cycle and apoptotic gene

expression. These findings in DVD-deficient embryonic brains are in accord with in vitro

studies in brain cells.(46,47) When the anatomy of DVD-deficient embryonic brains was

examined it was revealed that the newborn offspring of DVD-deficient rats had slightly

larger brains. This corresponded with an increased volume of the lateral ventricles and

a smaller neocortical width.(38) The structural brain phenotype of embryonic DVD-

deficient mice however would appear to be quite different with lateral ventricles being

shown to be reduced in the embryonic mouse brain.(44) This was also accompanied by

reduced brain length (44) rather than longer brains as seen in DVD-deficient newborn

rats.(38) Hippocampal volumes were also shown to be reduced in the DVD-deficient

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mouse but only in female newborns.(48) Many of these anatomical differences persist

into adulthood. The enlarged lateral ventricles seen in the DVD-deficient rat neonates
(38)
persists into adulthood if vitamin D deficiency is continued until weaning.(49) The

opposite finding regarding a decrease in lateral ventricles in mouse embryos also

appears to persist into adulthood.(48,50) Two separate groups have also now shown

DVD-deficiency in C57B6J mice produces a larger striatum and smaller hippocampus in

adult males.(48,50) The reason for why DVD-deficiency would induce contrasting brain

structural findings between species remains unknown. There may be a differential effect

of DVD-deficiency on brain cell proliferation between rats and mice but until this is

directly studied in mice this remains speculative. Vitamin D deficiency has also been

associated with a 28% increase in lateral ventricles in aged humans.(51)

Of all neurotransmitters to be linked with DVD-deficiency, dopamine is the one most

reported. DVD-deficiency may also adversely affect the ontogeny of other

neurotransmitter systems such as serotonin, however as far as we are aware, such

evidence remains only at the in vitro level.(52-54) As previously mentioned in developing

brains the VDR appears very early at embryonic day (E)12.(28,29) This represents the

peak age when most dopamine neurons are being born.(55) When mesencephalon was

harvested from vitamin D deficient embryos at this age we showed DVD-deficient

embryonic brains had a reduction in Nurr 1 and p57kip2a which are two crucial

specification factors for the maturation of DA neurons.(56) Genetically ablating these two

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factors leads to a reduction in DA cell number and altered positioning.(57-59) In a later

study we confirmed that early positioning of DA neurons in the developing

mesencephalon was indeed altered with an increase in laterally migrating DA neurons

in DVD-deficient brains.(60) We also measured dopamine levels in DVD-deficient

neonatal forebrain and showed that, although dopamine levels were normal, its

metabolism was altered with increased ratios of the two major dopamine metabolites,

3,4-dihydroxyphenylacetic acid/homovanilic acid (DOPAC/HVA).(61) This was

accompanied by a reduction in catechol-o-methyl transferase (COMT), the enzyme that

converts DOPAC to HVA.(61) Additionally, a recent study has shown gene expression

and protein content of the rate-limiting enzyme in DA production tyrosine hydroxylase

(TH) is reduced in DVD-deficient fetal mouse brains.(44) This same study has shown

DVD-deficiency in mice decreases the neurotrophin Brain-derived Neurotrophic factor

(BDNF) at early stages of brain development with a reversal at later stages.(44) This

study also showed DVD-deficiency decreased the expression of TGF-β1, an important

factor in dopaminergic differentiation. Enzymes involved in corticosterone metabolism

were also shown to be decreased.(62) Some DA abnormalities persist through to

adulthood with DA transporter density in the caudate putamen and DA binding affinity in

the nucleus accumbens both being increased in DVD-deficient adult rats.(63)

DVD-deficiency also has long-term effects on gene and protein expression in adult

brains. Gene array analysis on whole brain and proteomics in the prefrontal cortex and

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hippocampus of adult animals who were subjected to DVD-deficiency show alterations

in the expression of 74 genes and 36 proteins involved with such diverse functions as

cytoskeleton maintenance, calcium homeostasis, synaptic plasticity and

neurotransmission, oxidative phosphorylation, redox balance, protein transport,

chaperoning, cell cycle control and post-translational modifications.(64,65) A study of

protein expression in the nucleus accumbens of DVD-deficient rats showed that

although the degree of gene dysregulation was mild there were significant alterations in

several proteins involved in either calcium binding (calbindin, calretinin and hippocalcin),

or mitochondrial function.(66) One earlier study also showed DVD-deficiency induced

reductions in NGF, and neurofilament proteins indicative of delayed neuronal

maturation.(49)

IV. Developmental vitamin D (DVD)-deficiency and animal behavior

Behaviour of DVD-deficient rats

Alterations in maternal/pup interactions can produce long lasting changes in offspring

behaviours.(67) In particular, the quality of nursing behaviours, pup/dam communication

via pup ultrasonic vocalisations and how the dam retrieves pups once separated from

the main nest can all produce long-term effects in adult behaviour. Pup/Dam

interactions have recently been examined in DVD-deficient animals. Yates and co-

workers showed vitamin D-deficient dams exhibited decreased licking and grooming of

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their pups but no differences in pup retrieval. Perhaps consistent with this, DVD-

deficient pup ultrasonic vocalisations (a form of pup/Dam communication) was also

increased. As adults, males that had been exposed to vitamin D deficiency in early life

exhibited decreased social behaviour, impaired learning and memory outcomes and

increased grooming behaviour.(68) We have also examined many of these interactions in

DVD-deficient rats. Again, we show an increase in pup vocalisations (69) and similar

reductions in social interaction in offspring at later stages of development. We also

show some early climbing and self-righting reflex deficits indicative of delayed

development.(69) Additionally, these animals had impairments in normal ethologically

valid stereotyped digging behaviours. Many of these behaviours are considered

important phenotypes in animal models of relevance to autism.(70)

As adults, locomotion in response to a novel open field is enhanced in DVD-deficient

rats.(37) Locomotion in response to psychomimetic agents has also been assessed in

DVD-deficient rats. Using the N-methyl-D-aspartic acid receptor (NMDA-R) antagonist,

MK-801, an agent well-known to induce hyperlocomotion, adult male DVD-deficient rats

have been repeatedly shown to have enhanced locomotor activity compared to

controls.(40,71,72) This MK-801-induced hyperlocomotion in adult male and female DVD-

deficient rats is associated with a significant reduction in MK-801 binding in the caudate

putamen.(72) We have also shown that the later period of gestation was most relevant

period of DVD-deficiency for this effect.(40) This is reminiscent of our earlier findings

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regarding structural changes in the brains of these animals.(49) Therefore, it appears the

developmental window in which vitamin D deficiency is induced is also critical for

behavioral outcomes.

DVD-deficient rats were also selectively sensitive to the locomotor-enhancing effects of

amphetamine, a drug that induces presynaptic dopamine release.(63) A number of

studies have also shown that DVD-deficient rats are selectively sensitive to postsynaptic

dopamine blockade, in particular the dopamine 2 receptor blocker, haloperidol (which is

a widely used antipsychotic agent). The locomotor retarding effects of haloperidol

appeared to be greater in DVD-deficient animals when hyperlocomotion had first been

induced by MK-801.(71) In a separate study haloperidol was shown to normalize an

endogenous habituation deficit in DVD-deficient animals whereas it resulted in

habituation deficits if administered to control animals.(42) Using electrophysiological

recordings from the hippocampus of freely moving rats, a subsequent study investigated

long-term potentiation (LTP), which is a cellular correlate of learning and memory.(73) It

was shown that DVD-deficient rats had enhanced LTP, and this was reversed by

treatment with haloperidol. DVD-deficient rats also appeared to have normal pre-pulse

inhibition (PPI) (71) and working memory but disrupted latent inhibition, which is a

measure of attentional processing.(74) Although manipulating striatal dopamine release

can affect all of these three behaviors, this potential mechanism has not yet been

investigated in vivo.

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In a continuous performance task developed by Turner and colleagues DVD-deficiency

produced animals that had both increased premature responding, reflecting increased

impulsivity, and increased responding to non-target stimuli. Both behaviors indicate a

lack of response inhibition.(75) Importantly both of these behaviors were normalized with

acute treatment with the antipsychotic clozapine.(75) Finally we have recently shown

associative learning to be impaired in DVD-deficient rats.(76) Clearly there are multiple

behavioural abnormalities in these animals many of which are of potential relevance to

phenotypes of interest to schizophrenia and autism research.

Behaviour of DVD-deficient mice

DVD-deficiency in one mouse strain (129/SvJ) produced spontaneous hyperlocomotion

in the open field arena, similar to DVD-deficient rats.(71) Another widely used strain

C57BL/6J mice exposed to DVD-deficiency showed increased perseverative responses

to the target stimuli in a continuous performance task.(77) This indicates DVD-deficiency

induces similar deficits in response inhibition in both species. However all other

behavioural phenotypes of DVD-deficient mice are distinctly different from the rat.

DVD-deficient mice from both strains demonstrate an increased frequency of head dips

in a hole board arena, indicative of increased exploratory behavior.(78) This is in contrast

to findings from DVD-deficient rats on the same test.(42) Also the robust locomotor

response seen in DVD-deficient rats when exposed to MK-801 or amphetamine is not

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found in mice.(48) Another group have tested DVD-deficient C57BL/6J mice on a

hippocampal-dependent memory task known as the olfactory tubing maze. A learning

deficit was seen on the final day of training, with DVD-deficient mice showing a

reduction in the number of correct responses when compared to controls.(50)

Clearly the behavioral phenotype of the DVD-deficient rat is distinctly different to that of

the mouse. However, the array of behaviors examined indicate subtle alterations in

learning and memory in both species. The effects of DVD-deficiency on cognitive

function in children is far from clear. One study has shown children who were vitamin D

deficient during pregnancy had delayed cognitive development,(79) but this finding was

not replicated in a larger study when a broader array of cognitive outcomes was

assessed.(80) Whilst a comprehensive summary of the differences in brain structural and

behavioral outcomes in DVD-deficient rats and mice has recently been published,(81) an

exhaustive comparison between the effects of DVD-deficiency in both species has not

yet been conducted. Perhaps a more useful line of inquiry would be an examination of

which critical developmental window of exposure and which critical threshold of vitamin

D deficiency are required in order to change brain function in adult offspring.

To induce vitamin D deficient signaling via a genetic approach, groups have either

ablated the receptor (30,82-84) or the major synthetic enzyme for 1,25(OH)2D3

(CYP27B1).(85,86) In these models the VDR or enzyme are constitutively ablated.

Although VDR knockout mice have impairments on a range of behaviors or relevance to

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psychiatry such as anxiety,(87) neophobia,(88) altered nest building(89) they also have

severe phenotypes unrelated to brain such as hypertension and increased fluid

intake,(90) cardiac hypertrophy,(91) altered heart function,(92) impaired energy

metabolism,(93) musculoskeletal changes,(94) growth retardation, impaired motor

coordination and muscle fatigue. (95,96)

To date there are no published studies using conditional or brain specific VDR mutant

mice to test the effects of transient or regional receptor disruption on brain development.

Although tissue specific inactivation of CYP27B1 has been demonstrated (97) these

homozygous mutants also have a confounding rickets-like phenotype. These “off-target”

deficits complicate the interpretation of the genetic contribution of vitamin D signaling to

behavior.

V. Adult vitamin D (AVD)-deficiency/supplementation and brain and behavior

As previously mentioned, there are also numerous studies linking low vitamin D status

with schizophrenia, Alzheimer’s disease, dementias and Parkinson’s disease (for a

detailed summary of this literature see (13)). In order to investigate the biological nature

of these links, various preclinical models of adult vitamin D (AVD) deficiency have been

developed. One complicating factor in many earlier animal studies of AVD-deficiency

was the failure to address hypocalcemia which can radically affect brain function

therefore such studies will not be discussed further here.

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As cognition is impaired in most of the afore-mentioned disorders linked with AVD-

deficiency this behavior has been the most commonly assessed in AVD models

however the picture is far from clear. Six weeks of vitamin D deficiency is insufficient to

change cognitive responses in an AVD rat however it did lead to premature responses

indicating some effect on vigilance.(98) There were also small changes in striatal

neurotransmitter content including increased Gama-amino butyric acid (GABA) and

alterations in DA turnover. While much longer periods of vitamin D deficiency (6 - 12

months) increased reactive oxygen production in the brain (99) it also did not affect

cognition.(100) Mild cognitive deficits have been shown in some studies using AVD-

deficient mice (101) along with alterations in the major excitatory neurotransmitter in the

brain, glutamate and the major inhibitory transmitter GABA.(102) How these alterations

could directly relate to impaired cognition however is not immediately obvious. The

addition of high dose cholecalciferol (10 x normal dietary levels) did however appear to

increase memory outcomes in one study.(103)

In related studies AVD-deficiency has been shown to increase corticosterone response

to stressful events and increased avoidance times.(104) One specific learning task did

appear to be affected by AVD-deficiency with AVD-deficient rats having impairments in

aversive spatial learning. Importantly these findings were correlated with connectivity

abnormalities in the major brain region associated with spatial navigation, the

hippocampus.(105)

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In respect to Alzheimer’s disease (AD) there are numerous transgenic models

mimicking the brain pathology of the disease. Given the ongoing reports of vitamin D

deficiency in patients with AD a number of studies have been initiated to see if cognitive

decline and or brain toxicity could be attenuated by vitamin D treatment in such models.

These studies have mostly supported the idea that vitamin D is neuroprotective. For

instance vitamin D enriched foods decrease brain pathology and prevent cognitive

decline.(106) These findings were largely replicated by dietary supplementation.(107)

Similar outcomes were found using acute exposure to 1,25(OH)2D3 (108) with the

hormonal form of vitamin D also appearing to increase elimination of pathological ß-

Amyloid proteins from the brain.(109,110) Models of dietary insufficiency have also shown

to lead to greater AD-related brain pathology.(107,111)

Similar to the situation of AD there are numerous genetic or toxin-based models of

Parkinson’s disease (PD). Again disease-specific pathology would appear to be

alleviated by acute administration of the active hormone 1,25(OH)2D3.(112-115) This would

appear to be via upregulation of the rate-limiting enzyme for DA production Tyrosine

Hydroxylase (TH).(116,117) We have now outlined the direct mechanisms for how vitamin

D regulates TH gene expression (117,118) (and see below). 1,25(OH)2D3 also ameliorates

the oxidative burden many of these PD models induce in brain.(119)

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VI Vitamin D regulates essential processes in normal brain development and

function and is neuroprotective in adult brains

So far, we have outlined how vitamin D deficiency may adversely affect essential

normal processes in brain development, adult brain function and behaviour. In this next

section we review the basic evidence for how vitamin D exerts influence over crucial

events in brain ontogeny such as axonal elongation, neurotrophin production,

neurotransmitter synthesis and how it can act to protect neurons from a range of

adverse exposures.

A. Vitamin D and axonal growth

We were the first group to show the addition of 1,25(OH)2D3 to embryonic hippocampal

explant cultures increased neurite outgrowth.(46) This finding was replicated more

recently in individual hippocampal neurons using the same concentration of

1,25(OH)2D3.(47) Both groups described a small but significant elevation in NGF and

assumed this effect was causal. Another group chose to examine the ability of

ergocalciferol (Vitamin D2) to enhance axon regeneration after peripheral denervation.

These authors chose ergocalciferol based on an older study that claimed this was a

more potent form than cholecalciferol in elevating 25OHD2 levels in rats.(120) These

authors were able to demonstrate increased axiogenesis, axon diameter and higher

functional recovery if ergocalciferol treatment was initiated immediately after

lesioning.(121) We also now have new unpublished data replicating the neurite promoting

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potential of 1,25(OH)2D3 in developing DA neurons differentiated from a) a

neuroblastoma cell line, b) primary mesencephalic DA neurons and c) explant

mesencephalic cultures. We conclude that like all other neurosteroids, vitamin D

enhances neurite extension. We are now exploring the molecular mechanisms for these

effects.

B. Vitamin D and Neurotrophic factors

Vitamin D’s actions in promoting neurotrophic factors such as NT-3, NT-4 (122) and nerve

growth factor (NGF) in particular (46,47,122-124) have been well described. NGF is

particularly important in the development and survival of hippocampal neurons in either

cultured explants (46) or in individual cultured cortical neurons.(125) Silencing VDR

expression leads to a corresponding reduction in NGF production in primary cortical

neurons.(126) Administration of 1,25(OH)2D3 directly to the hippocampus of adult rats

also induces NGF expression. Therefore the evidence that vitamin D may be required

for ongoing neuronal survival in adult brains via such mechanisms appears strong.(127)

Given its role in dopaminergic neuron differentiation and survival there has also been a

strong focus on vitamin D and neurotrophic factors specific to dopaminergic neurons

such as glial derived neurotrophic factor (GDNF) (128,129) and brain-derived neurotrophic

factor (BDNF) again for its broad trophic actions in the developing and adult brain.

Neural stem cells treated with 1,25(OH)2D3 show increased expression of NT-3, BDNF

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and GDNF.(130) Cultured mesencephalic neurons which contain most of the developing

DA neurons in the brain increase GDNF expression after 1,25(OH)2D3 administration

with an increase in DA cell number also. This vitamin D mediated increase is blocked

when GDNF synthesis is chemically blocked.(131) We have recently shown direct

genomic evidence for how vitamin D directly regulates the transcription of both

receptors for GDNF. 1,25(OH)2D3 suppresses GDNF family receptor alpha 1 (GFRa1)

but the liganded VDR directly binds to the promoter of the proto-oncogene tyrosine–

protein kinase receptor Ret (C-Ret) which is the other major receptor for this

neurotrophin, to upregulate C-Ret expression. Correspondingly the maternal absence of

vitamin D decreases C-Ret expression in the developing rat mesencephalon.(118)

Very few studies to date have examined the effect of vitamin D on these neurotrophic

factors in the developing brain. In the studies that have, one showed DVD-deficiency in

rats induced deficits in neonatal whole brain NGF and GDNF protein.(38) A much more

recent study also showed early reductions in BDNF and transforming growth factor-β1

(Tgf-β1) in DVD-deficient embryonic mouse brains.(44)

Almost universally, the breadth of work over the past two decades indicates

1,25(OH)2D3 increases, and the absence of vitamin D in the maternal diet reduces the

expression of these important neurotrophic factors in neurons and glia of developing

brains. These factors remain highly attractive candidate pathways in understanding the

role vitamin D plays in brain ontogeny.

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C. Vitamin D as a regulator of dopamine in development

1,25(OH)2D3 has been shown to alter cholinergic, dopaminergic and noradrenergic

neurotransmitter systems in vitro.(132,133) Our data to date in DVD-deficient brains

strongly and consistently indicates the absence of this vitamin during fetal brain

development appears to produce adverse effects on developing and to a lesser extent

adult, dopamine systems. We were the first group to report intense

immunohistochemical staining of the VDR within Tyrosine Hydroxylase (TH) positive

neurons within the human substantia nigra.(3) Since then we have confirmed that TH

positive neurons in the neuromelanin containing human nigra are VDR positive and we

have now mapped the ontogeny of VDR expression in rat brain.(28) As previously

discussed, the absence of vitamin D during development decreases the expression of

crucial specification factors for dopamine neurons (56) and reduces enzymes involved in

DA turnover with accompanying alterations in DA metabolites.(61) We have recently

shown DVD-deficiency alters the positioning of the 2 major DA neuron clusters in

embryonic brains with an imbalance between DA neurons in the substantia nigra

compared with the ventral tegmentum along with reductions in the expression of Nurr1

and TH indicative of a delay in DA neuron differentiation.(60) In a later study we showed

1,25(OH)2D3 was capable of rescuing deficits in DA specification factor expression, DA

progenitor cell number and positioning abnormalities in DA neurons induced by

maternal immune activation.(134)

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We have confirmed that 1,25(OH)2D3 positively regulates TH mRNA and protein and the

metabolic product of TH, dopamine using a VDR-over-expressing neuroblastoma cell

system.(117) Simply increasing VDR expression alone in the absence of 1,25(OH)2D3 is

also sufficient to drive undifferentiated cells down a dopaminergic lineage.(135) In

addition we have established that 1,25(OH)2D3 increases VDR regulation of a major

metabolic enzyme for DA in the brain, Catechol-o-methyl transferase (COMT).

Chromatin immuno-precipitation data confirm the liganded VDR binds to the COMT

promoter strongly suggesting a direct regulation of COMT gene expression.(135) Another

group has shown 1,25(OH)2D3 may drive TH and therefore DA production via a GDNF-

mediated mechanism.(131) We and others, are now engaged in trying to understand how

such early changes in the formation of dopaminergic systems could affect downstream

brain function in mature animals.(136) 1,25(OH)2D3 has also been administered to

newborn rats and dopamine and noradrenalin measured in a variety of brain regions in

these animals as adults. These authors showed that dopamine and noradrenalin were

elevated mainly in the brainstem of these animals as adults.(137)

Considered in its totality, the consistent findings of impaired DA neuron maturation in

DVD-deficient embryonic brains, impairments in behavior influenced by dopamine in

DVD-deficient adults, coupled with our most recent data showing vitamin D signaling in

cultured neurons drives neuron maturation down a dopaminergic lineage, all combine to

strongly suggest vitamin D plays a crucial role in the early ontogeny of dopamine

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systems. Given we have established that DVD-deficiency is a developmental

epidemiological risk factor for schizophrenia (8,9) and that dopamine abnormalities are

also strongly linked with this disease, these data may prove important for the etiology of

psychiatric disease.

D. Vitamin D and possible neuroprotective mechanisms in the developing and adult

brain

Here we focus on three common exposures, excessive calcium, reactive oxygen

species (ROS) and corticosterone which are all naturally occurring in the brain but when

are elevated experimentally and by analogy in various disease states, are known to

adversely affect brain function and in some cases lead to long-term pathology. These

exposures are all exacerbated by vitamin D deficiency and ameliorated by the addition

of 1,25(OH)2D3 or dietary cholecalciferol.

Calcium transients are essential for normal neuronal function, however unbuffered

calcium is neurotoxic for brain cells. It is well known how vitamin D regulates calcium

uptake in non-neuronal cells such as osteoblasts and osteosarcoma cells via direct

regulation of calcium channels.(138,139) Now the actions of vitamin D are being studied in

neurons and brain. Studies in vitro show 1,25(OH)2D3 blocks calcium influx and

therefore toxicity in cultured mesencephalic neurons (140) or hippocampal neurons

(141,142)
via the down-regulation of L-type voltage-sensitive calcium channels.(126)

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Silencing VDR expression blocks this.(126) However the rapid non-genomic actions of

vitamin D produce the opposite effect with an increase calcium influx in cortical slices

which is again dependent on L-type calcium channel activity.(143) Recently we have used

calcium imaging, electrophysiology and molecular biology to further explore the non-

genomic actions of 1,25(OH)2D3 on cortical neurons. We show physiological

concentrations of 1,25(OH)2D3 rapidly enhance calcium influx but only in a small subset

of neurons. Somatic nucleated patch recordings revealed a rapid, 1,25(OH)2D3-evoked

increase in high-voltage-activated calcium currents and again these were mediated by

L-type voltage-gated calcium channels.(144) Examination of the function of vitamin D

signaling on the activity of these channels is worth further scrutiny given the close links

between genetic variants in these channels and schizophrenia.(145)

The anti-oxidant potential of vitamin D in a variety of tissues including isolated neurons

(140,146)
and brain (114,147) has long been known. In general this is believed to be due to

the ability of vitamin D to increase potent anti-oxidant molecules such as glutathione

and cytochrome c. A very recent study showed AVD-deficiency increased reactive

oxygen species and extracellular calcium in the brain. This occurred along with

impairments in GABA and Glutamate release. Importantly all deficits were normalized

by reintroducing dietary vitamin D.(148)

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One long-speculated neuroprotective mechanism of 1,25(OH)2D3 in the brain has been

the potential inhibition of Nitric Oxide (NO) production.(4) In the brain, microglia are the

immunologically responsive cells responsible for the production of inflammatory

regulators such as NO. Early reports suggested vitamin D could affect

neuroinflammation and microglial activation. 1,25(OH)2D3 inhibits the expression of

inducible nitric oxide synthetase in the rat brain during either experimental allergic

encephalomyelitis (149) or after intracranial injection of the lipopolysaccharide endotoxin

(LPS).(150) 1,25(OH)2D3 also reduces the production of proinflammatory cytokines and

NO in microglial cells.(151) Later studies have focused on potential molecular

mechanisms. Microglia when activated with LPS increase production of CYP27B1 and

as a result 1,25(OH)2D3. In an important study, when LPS-induced elevation of NO was

examined in cultured microglia, the addition of 25(OH)D3 reduced NO production

presumably via local synthesis of the active hormone 1,25(OH)2D3. Confirmation of this

came from treating these same microglia with silencing RNA directed against CYP27B1

which reversed the inhibitory effect of 25(OH)D3.(152) Treatment with 1,25(OH)2D3 also

attenuates LPS-induced ROS production, NO accumulation, and inducible nitric oxide

synthase expression in concentration-dependent manners in primary cortical neurons in

culture.(153) Finally a very recent study in synaptosome preparations from vitamin D

deficient adult rats showed increased ROS and higher calcium influx indicating

increased excitability. Importantly this was reversed in rats in which vitamin D deficiency

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had been corrected by supplementation.(148) In summary, microglia would appear to

represent the site for the anti-oxidant actions of vitamin D in the brain.

The secretion of glucocorticoids is a classic endocrine response to stress. Prolonged

exposure to increased levels of this hormone induces neuronal atrophy and eventually

cell death.(154) In general the cellular effects of 1,25(OH)2D3 and glucocorticoids are

considered to be antagonistic.(155-159) This would also appear to be the case in the brain

with 1,25(OH)2D3 antagonizing the effects of the corticosterone agonist dexamethasone,

on hippocampal neuron differentiation and glucocorticoid receptor function.(160) This

process is reversible with dexamethasone shown to decrease the expression of

enzymes involved in both the synthesis and turnover of 1,25(OH)2D3 in the

hippocampus and prefrontal cortex.(161)

The antagonism between corticosterone and vitamin D would also appear to be

reflected at a behavioural level. Chronic cortisol administration induces depression-like

phenotypes in animals and in a remarkably consistent pattern vitamin D would appear

to ameliorate or completely reverse this. In terms of adult behavior, vitamin D reverses

depressive behavioural phenotypes induced by chronic corticosterone.(162-165) Putative

mechanisms include regulation of glucocorticoid receptor expression in hippocampus or

via restoring DA levels in the reward centres in the brain.

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DVD-deficiency may also alter maternal stress responsivity in both rats (166) and mice.(62)

DVD-deficiency also adversely affects maternal care (68) which as previously outlined

can also produce long-lasting changes in stress-responsivity in offspring.(67)

VII. Conclusions

In this mini-review we have concentrated on summarising the pre-clinical literature

outlining vitamin D metabolism, genomic and non-genomic signalling in brain. We urge

caution when interpreting previous constitutive knock out strategies to genetically alter

vitamin D signalling given the vast array of non-brain related alterations they induce.

Similarly, the older dietary manipulations frequently induced hypercalcaemia which

would obscure any individual contribution of vitamin D to brain function. In addition,

there are also clear species and even strain differences in respect to the dietary effects

of vitamin D deficiency in rodents. The current DVD- or AVD-deficient rodent models

have no such impediments producing animals which are normocalceamic and appear

physiologically normal. The breadth of data obtained from such models confirm vitamin

D as an important neurosteroid for both developing and adult brains producing animals

in which there are abnormalities in a diverse range of behavioural phenotypes of

interest to both psychiatry and neurology.

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Epidemiological studies linking low neonatal vitamin D with disorders of brain

development such as autism and schizophrenia continue to emerge. Similarly, the link

between low levels of vitamin D and degenerative conditions such as AD and PD is

progressively strengthening. As a result, speculation continues to be made as to the

relevance of adequate vitamin D levels in the possible prevention/amelioration of such

disorders. This of course would be difficult to test directly for developmental conditions

with an adult onset such as schizophrenia and therefore this association can only be

made retrospectively. However, for childhood onset psychiatric conditions and ongoing

degenerative conditions, properly designed randomized clinical trials of vitamin D

supplementation in such risk groups are likely to yield interpretable data in a timely

fashion.

We again would like to insert a note of caution in the light of certain recent high-profile

reports. Unfortunately in many observational epidemiological studies of vitamin D status

and psychiatric outcomes the issue of reverse causality (the condition induces low

levels of vitamin D rather than the reverse) is often not, or poorly addressed. This is

made all the more relevant give a very high-profile recent report in the New England

Journal of Medicine showing virtually all mental illnesses were associated with an

increased risk of a subsequent non-psychiatric medical condition.(167) This has

significant implications for psychiatric research in general. It is also highly relevant to

any proposed association between low vitamin D levels reported in adults with any

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psychiatric or neurological condition as sick people are probably not looking after their

diet or getting adequate exercise and exposure to sunshine. This is of perhaps

diminished relevance to conditions associated with DVD-deficiency. We urge all future

epidemiological studies that seek to examine the relationship between vitamin D and

psychiatric or neurological conditions to rigorously control for the established poor

general health of patients with psychiatric conditions.

It is also salient to mention another recent landmark study that used mendelian

randomization models to examine gene pathways related to 25(OH)D3 blood

concentrations. This study could find no evidence that genetic factors involved in the

production of 25(OH)D3 were causal for psychiatric disorders.(168) To us this suggests

any link between 25(OH)D3 levels and any brain related outcome are likely to be solely

driven by environmental factors .

By now there has been sufficient interest in the links between vitamin D and brain-

related disorders for contrary findings to begin to emerge. For instance, it is illustrative

to examine a number of recent publications showing a predicted inverse relationship

between DVD-deficiency and autism.(10-12,169) These studies all had mean 25(OH)D3

levels of <50nM which is considered by some authors to represent a cut off for vitamin

D deficiency. Two recent studies have failed to find this inverse association.(170,171) So,

at face value, this appears a failure to replicate previous studies. However, it is crucial

to note that in these last two studies, the mean levels of 25(OH)D3 were actually very

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high (>70-80 nM) and there were very few individuals that were actually vitamin D

deficient meaning the association could not be properly addressed. These same six

studies all used the same laboratory to analyse samples so technical bias (so common

amongst vitamin D studies in different populations) could be ruled out. This suggests a

threshold effect rather than any continuous relationship between DVD-deficiency and

autism. We highlight this particular relationship to illustrate some of the confusion

regarding statements regarding potential causality between vitamin D and various brain-

related clinical disorders.

We believe that if all such possible confounds can be carefully considered in the future,

then more clarity might be brought to the next generation of epidemiological

investigations examining vitamin D levels in psychiatric or neurological conditions. It

remains an extremely attractive option to use such a simple, safe and inexpensive

intervention to alleviate the substantial disease burden these conditions carry for

patients. Given the alarming prevalence of hypovitaminosis D in both pregnant women

and their newborns (172) and in the general population, ensuring the diverse functional

capacities of this neuroactive steroid in the developing and adult brain are preserved

through either environmental or dietary interventions would appear to be a vital public

health priority. Ultimately only well-designed randomized double blinded clinical trials

will reveal the therapeutic relevance of vitamin D in brain-related disorders.

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Figure Legend

Figure 1. Vitamin D and its effects on brain and behavior. A) Depicts the progressive

molecular, cellular, brain structural and behavioural abnormalities induced in the DVD-

deficient rat model. B) Far less investigation has been conducted on AVD-deficiency

reflecting less certainty regarding the use of this model to study brain disorders. C)

There have however been numerous studies in models of relevance to either

Alzheimer’s or Parkinson’s disease indicating intervention with vitamin D may have

therapeutic potential.

Acknowledgements This research receives funding from the National Health and

Medical Research Council (APPS 1124721 and 1141699) and the Queensland State

Government. We thank Suzy Alexander for assistance creating the figure.

Conflicts. The authors have no conflicts of interest

Author contribution DE conceived and wrote the manuscript

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Accepted Article

Figure 1. Vitamin D and its effects on brain and behavior. A) Depicts the progressive molecular, cellular,
brain structural and behavioural abnormalities induced in the DVD-deficient rat model. B) Far less
investigation has been conducted on AVD-deficiency reflecting less certainty regarding the use of this model
to study brain disorders. C) There have however been numerous studies in models of relevance to either
Alzheimer’s or Parkinson’s disease indicating intervention with vitamin D may have therapeutic potential.

296x209mm (300 x 300 DPI)

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