The Origins of Schizophrenia

It is widely accepted that schizophrenia is a heterogeneous group of disorders influenced by a variety of genetic and environmental factors. Researchers have explored the association between schizophrenia and a range of candidate early life exposures that could disrupt early brain development (e.g., prenatal stress, malnutrition, infection, and obstetric complications). On the basis of the clues from epidemiology, our group proposed that low prenatal vitamin D₃ is a modifying risk factor for schizophrenia. In 1999, we developed an informative animal model—the DVD model—in which vitamin D₃ is depleted in utero. The DVD-deficient offspring reproduce several features observed in patients with schizophrenia, such as enlarged lateral ventricles and altered behavior in response to psychomimetic agents. In this chapter, we integrate findings derived from the DVD model. The first section concisely summarizes clues from the epidemiology of schizophrenia. The second section discusses the evidence regarding the biological importance of vitamin D₃ in brain development and function. The third section reviews the experimental findings in DVD rodent models, showing that maternal vitamin D₃ deficiency can lead to long-lasting neuroanatomic, neurochemical, and behavioral changes that are relevant to the disease. The final section outlines future research directions and the paradigm for testing this hypothesis in human populations.

Prenatal Low Vitamin D₃ as a Modifying Risk Factor for Schizophrenia: Clues from Epidemiology

The prenatal hypovitaminosis D₃ hypothesis was inspired by epidemiologic evidence. One of the most consistently replicated epidemiologic features of schizophrenia is the slight but significant excess of individuals with schizophrenia who were born in winter or spring months as opposed to other months of the year (Bradbury & Miller, 1985; Torrey et al., 1997; Torrey & Miller, 1997), and this feature is more prominent at high-latitude sites (Davies et al., 2003; Saha et al., 2006). Results from a large population-based Danish study showed that there was a small seasonal excess of schizophrenia births in winter and spring months (relative risk = 1.1) (Mortensen et al., 1999). Another interesting finding from this study was that the relative risk of developing schizophrenia was about 2.4-fold higher for those born in the city than for those born in the country. Other groups have also identified an excess of schizophrenia births in urban centers (Marcelis et al., 1998; Mortensen et al., 1999; Pedersen & Mortensen, 2001). Moreover, the incidence of schizophrenia is significantly higher in dark-skinned migrants to cold countries than in the native-born of those countries (Cantor-Graae, Zolkowska, & McNeil, 2005). Low prenatal vitamin D₃ “fits” these key environmental features: (1) vitamin D₃ deficiency is common during winter and at high latitude, most likely because of an decrease in sunlight duration, sunlight intensity, and outdoor activity (Holick, Matsuoka, & Wortsman, 1995); (2) city dwellers tend to have less exposure to sunlight and thus have lower 25-hydroxyvitamin D₃ (25[OH]D₃) levels (McGrath et al., 2001); and (3) hypovitaminosis D₃ is more common in dark-skinned populations (Holick, Matsuoka, & Wortsman, 1995).

In addition to these observations from ecologic epidemiology, preliminary evidence from a case-control study provides some support for the hypothesis. The 25(OH)D₃ serum levels in 26 mothers whose children developed schizophrenia were numerically (but not significantly) lower than those of 51 mothers whose children did not develop the disease (McGrath et al., 2003). In a subsample of African Americans (seven cases and 14 controls), a trend level association (p = 0.08) was found between low maternal vitamin D₃ levels and offspring who developed schizophrenia. Furthermore, vitamin D₃ supplements in the first year of life significantly reduced the risk of schizophrenia in males in a large Finnish birth cohort (McGrath et al., 2004).

Vitamin D₃ has long been known to control blood levels of calcium by regulating genes involved in calcium intestinal absorption, renal excretion, and movement in and out of bone (Heaney, 2007). In recent years, an ever-widening range of vitamin D₃ actions has been described, including regulation of proliferative and apoptotic activity (Reichrath et al., 2007), immunomodulation (Bouillon et al., 1995; Deluca & Cantorna, 2001), and neuroprotection (Garcion et al., 2002; Holick, 2007; McCann & Ames, 2008). In humans, vitamin D₃ is produced after ultraviolet B (UVB) light photolyzes 7-dehydrocholesterol in the skin, forming previtamin D₃ (Holick, 1988) (figure 11.1). The liver and other tissues metabolize vitamin D₃ to 25(OH)D₃, the principal circulating form of vitamin D₃. This intermediate is then further hydroxylated by 1α-hydroxylase (1α-OHase) in the kidney to 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃), the biologically active form of the vitamin. Although some vitamin D₃ can be obtained from the diet, the majority of circulating vitamin D₃ is obtained from the action of sunlight on the skin.

1, 25(OH)₂D₃, the active form of the vitamin D₃, can cross the blood-brain barrier (Pardridge, Sakiyama, & Coty, 1985). The concentration of 1,25(OH)₂D₃ in normal adults has been reported as 31 pg/ml (range: 10–55) in serum and 25 pg/ml (range: 2–39) in cerebrospinal fluid (Balabanova et al., 1984). In addition, 1,25(OH)₂D₃ can be synthesized locally in the brain. Neuronal and glial cells can absorb circulating 25(OH)D₃ (Gascon-Barre & Huet, 1983) and produce 1,25(OH)₂D₃ by action of 1α-OHase. 1α-OHase has been detected in the fetal (Fu et al., 1997) and the adult human brain (Eyles et al., 2005), where it is distributed in both neurons and glia in a regionally and layer-specific pattern (Eyles et al., 2005). The strongest immunohistochemical staining of 1α-OHase is in the cytoplasm of neurons in the hypothalamus and the large (presumably dopaminergic) neurons in the substantia nigra. This catalytic enzyme is also found in cerebellar Purkinje cells and neurons in the cerebral cortex (Zehnder et al., 2001). In addition, microglia in culture can metabolize 25(OH)D₃ to produce biologically active 1,25(OH)₂D₃ (Neveu et al., 1994).

In addition to the presence of the active vitamin D₃ and its catalyzing enzyme, the vitamin D receptor (VDR) has also been identified in the human brain (Sutherland et al., 1992; Zehnder et al., 2001; Eyles et al., 2005). The VDR is expressed in cortical and noncortical regions, including hippocampal CA1, CA2, and CA3; the caudate-putamen; the thalamus; the hypothalamus; and the cerebellum. The distribution of the VDR and the fact that it can be synthesized locally is consistent with the proposal that vitamin D operates in a fashion similar to other neurosteroids (McGrath, Feron, & Eyles, 2001).

Figure 11.1. Vitamin D metabolic pathway.
In humans, although some vitamin D₃ can be obtained from the diet, the majority of circulating vitamin D₃ is obtained from the action of sunlight on the skin. Vitamin D₃ is produced after ultraviolet B (UVB) light photolyzes 7-dehydrocholesterol in the skin, forming previtamin D₃. The liver and other tissues metabolize vitamin D₃ to 25(OH)D₃, the principal circulating form of vitamin D₃. This intermediate is then further hydroxylated by 1α-hydroxylase (1α-OHase) in the kidney to 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃), the biologically active form of the vitamin. (Source: Authors.)

In the rat brain, VDR expression is developmentally regulated. Its distribution is prominent within the neuroepithelium and within the differentiating fields of various areas of the brain from embryonic days 12 to 21 (Veenstra et al., 1998). Using quantitative methods, our group found that the expression of VDR mRNA and protein increased markedly between embryonic days 17 and 19, a period that correlates with the increase in apoptosis and decrease in mitosis (Burkert, McGrath, & Eyles, 2003).

The VDR is also found in the subventricular zone (SVZ), one of the major sources of neural stem cells, and this expression is particularly prominent at birth (Cui et al., 2007). The temporal distribution pattern of VDR suggests that vitamin D₃ may influence cell differentiation in the developing brain. In addition, the influence of vitamin D has been intensively investigated in many types of cancer cells and other organs (Harrison, Wang, & Studzinski, 1999; Lin & White, 2004; Raiten & Picciano, 2004; Nagpal, Na, & Rathnachalam, 2005; Ebert et al., 2006). Our group has demonstrated that the addition of vitamin D₃ inhibits proliferation of neural progenitor cells isolated from the neonatal SVZ (Cui et al., 2007) and reduces mitotic cell number in embryonic hippocampal explants (Brown et al., 2003). In normal nervous system development, neurons are generated in numbers exceeding those found in adulthood. The surplus neurons are eliminated by programmed cell death, a process that is influenced by a range of external factors. The balance of proliferation and programmed cell death is critical for the orderly development of the brain. The effects of vitamin D₃ on neural cell number in vitro suggest a role in this critical aspect of brain development.

Taken together, several lines of evidence suggest that 1,25(OH)₂D₃ may influence brain development and that low maternal vitamin D₃ may be an environmental risk factor for schizophrenia. A decade ago, our group developed an informative animal model—the DVD model—in which vitamin D₃ is depleted in utero. This model displays a range of subtle but informative neurochemical and behavioral features in common with the clinical phenotype of schizophrenia. In the next section, we outline the findings from this animal model with respect to brain development, brain structure, and behavior.

To produce the DVD-deficient model, female Sprague-Dawley rats are fed a diet that lacks vitamin D but contains normal calcium and phosphorous. The rats are housed under a 12-hour light-and-dark cycle using incandescent lighting free of UV radiation in the vitamin D₃ action spectrum (290–315 nm). After six weeks, serum vitamin D₃ depletion is confirmed before mating using a verified in-house LC/MS/MS assay (25(OH)D₃ < 0.34 ng/ml) (Eyles et al., 2009). The resulting dams are housed under these conditions until the birth of pups. Control animals are kept under standard lighting conditions and are supplied with standard rat chow containing vitamin D₃. All dams (both control and depleted) are kept under standard housing conditions (control rat chow and UV-emitting lighting) after giving birth. Both vitamin D_3-depleted dams and offspring remain normocalcemic. It is important to stress that the exposure to vitamin D₃ depletion is only transient. DVD-deficient offspring become vitamin D₃ replete by two weeks of age and have normal levels of vitamin D₃, calcium, and phosphorous in adulthood.

The absence of maternal vitamin D has multiple effects on the developing brain. First, at the gross architectural level, DVD-deficient pups had cerebral hemispheres that were longer but not wider than controls. When corrected for hemispheric volume, these pups also had lateral ventricles that were larger than those of controls. In addition, after the data were normalized for whole-brain cross-sectional area, the neocortex of these pups was thinner than controls (Eyles et al., 2003).

Second, DVD deficiency increased mitosis globally across the embryonic brain. Consistent with the elevated cell division, the number of apoptotic cells was reduced in most brain regions from embryonic day 21 until birth embryonic day 23 (Ko et al., 2004). When neurosphere cultures were made from the brains of DVD-deficient neonates, neurosphere number was increased, suggesting greater rates of cell division (Cui et al., 2007). These results suggest that vitamin D exerts a pro-differentiation and proapoptotic function in the developing brain. Given that normal brain development requires precise spatial and temporal regulation of both cell proliferation and elimination, we speculate that the altered brain size and shape in DVD-deficient offspring may be the result of the disrupted normal progression of proliferation and cell death.

Low maternal vitamin D₃ also altered the gene expression profile regulating mitosis and apoptosis in the brain (Ko et al., 2004). Results from pathway-specific arrays showed that 74% of expressed apoptotic genes were down-regulated and 48% of genes related to mitosis were up-regulated in DVD-deficient pups. For example, at perinatal stages the expression of Bak, a pro-apoptotic gene, was decreased whereas cyclins A1, D1, and E, genes favoring progression of the cell cycle, were increased (Ko et al., 2004). Consistent with this pattern, both cyclin C and B, which are up-regulated by vitamin D₃ (Harrison, Wang, & Studzinski, 1999; Polly et al., 2000), were down-regulated in DVD-deficient rats. The cyclin-dependent kinase inhibitor p21cip1, which is up-regulated by vitamin D₃ in in vitro cell lines (Liu et al., 1996; Hager et al., 2001), was down-regulated in the DVD-deficient neonatal rat brain (Ko et al., 2004). In sum, at both cellular and transcriptional levels, the absence of vitamin D₃ disrupts the normal sequence of mitotic and apoptotic activity in brain development. This might explain the alterations in neonatal brain shape induced by DVD deficiency.

Apart from their role in synaptic conduction, neurotransmitters can act as trophic factors that regulate morphogenetic events such as proliferation, migration, differentiation, neurite growth, and neural circuit formation during brain development (Nguyen et al., 2001; Ruediger & Bolz, 2007). Therefore, we also examined whether neurotransmitter turnover, specifically dopamine (DA), is altered in neonatal brains. We showed that DVD deficiency induces a 45% reduction in the expression of catechol-O-methyl transferase (COMT), one of the key enzymes for DA metabolism in the cortex (Kesby et al., 2009a) (figure 11.2). Although the levels of DA or DA metabolites were not altered in these brains, the ratio of the DA metabolites—3,4-dihydroxyphenylacetic acid (DOPAC) to homovanillic acid (HVA)—was significantly greater in the forebrain, reflecting decreased conversion of DOPAC to HVA by COMT. The primary route of DA metabolism is through the intraneural oxidative deamination of DA to DOPAC by monoamine oxidase. DOPAC is then O-methylated extra-neuronally by COMT to HVA. Under normal conditions within the striatum, approximately 70–80% of HVA is produced by means of this pathway (Westerink, 1985; Wood, Kim, & Marien, 1987).

Figure 11.2. Key metabolic pathways related to dopamine neurotransmission.
The primary route of DA metabolism is through the intraneural oxidative deamination of 3,4-dihydroxyphenylacetic acid (DOPAC) by monoamine oxidase (MAO-A). DOPAC is then O-methylated extra-neuronally by catechol-O-methyl transferase (COMT), to homovanillic acid (HVA). The vesicular monoamine transporter (VMAT) avidly sequesters intracellular DA into vesicles. The vesicles are maintained within the neuron until a sufficient potential induces release at the synaptic membrane. Once released the DAT actively transports DA back into the neuron to cease signaling and allow for repacking of DA into the vesicles by VMAT. (Source: Authors.)

In summary, our findings confirm that DVD deficiency is capable of altering aspects of brain structure, cell proliferation, apoptosis, and neurotransmission during development. In the next section, we discuss abnormalities that persist in the adult brains of DVD-deficient animals and how these might relate to the structural, neurochemical, and behavioral alterations seen in patients with schizophrenia.

Depending on the timing of reintroduction of vitamin D₃ , the DVD model also exhibits persistent changes in adult brain outcomes. Although DVD deficiency during gestation yields marginal changes in lateral ventricle volumes in adult offspring, the volume is significantly increased if the introduction of the vitamin D₃-replete diet is delayed until weaning. A proteomics study of the frontal cortex and hippocampus from these animals revealed that DVD deficiency induced a reduction in proteins directly involved with cytoskeleton maintenance such as ß tubulin and the light and medium neurofilament proteins (Almeras et al., 2007). These changes may be relevant for the structural alterations seen in the adult brains.

DVD deficiency also altered proteins involved in mitochondrial function and neurotransmission in the nucleus accumbens (Nac) (McGrath et al., 2008). Nac, one of the projection regions in the mesolimbic DA system, is of particular interest with respect to schizophrenia (Lauer, Senitz, & Beckmann, 2001). DVD deficiency was associated with alteration in 35 unique proteins. Of these, 22 were down-regulated and 13 up-regulated. Six mitochondrial proteins were down-regulated in DVD-deficient rats (NDUAA, UQCR1, ODPB, IDH3A, HKK1, VDAC2). Neurotransmssion-associated proteins were also dysregulated. Two members of the dynamin family (dynamin 1 and dynamin 1-like proteins) as well as syntaxinbinding protein, which are involved in neurotransmitter vesicle release, were significantly down-regulated in the adult DVD-deficient rats. Apart from these proteins, four calcium-binding proteins (calbindin, calbindin2, hippocalcin, and calreticulin) were significantly altered in the Nac of the adult DVD-deficient rat. Calbindin, an important component of a subset of GABAergic interneurons (those that transmit or secrete gamma-aminobutyric acid), is strongly induced by vitamin D₃ (Christakos et al., 2007), and thus it is feasible that the reduction in this protein may be a direct consequence of the early life reduction in vitamin D₃.

Vitamin D₃ depletion in utero also had significant effects on the expression of genes involved in cytoskeleton maintenance (MAP2, NF-L) and neurotransmission (GABA-Aα4) (Feron et al., 2005). In the frontal cortex and hippocampus (Eyles et al., 2007), the expression of 36 genes involved in oxidative phosphorylation, redox balance, cytoskeleton maintenance, calcium homeostasis, synaptic plasticity, neurotransmission, and chaperoning was altered. With respect to human psychiatric disorders, many of these genes have been shown to be dysregulated in schizophrenia. Most prominent among these is the dysregulation of mitochondrial proteins. In schizophrenia, adenosine triphosphate (ATP) production in the frontal and left temporal lobes is reduced (Fujimoto et al., 1992; Volz et al., 2000). It is important that a genomic-proteomic study of frontal lobes from patients with schizophrenia revealed that genes connected to mitochondria function were among the most dysregulated (Lu et al., 2000). Subjects with schizophrenia display dysregulated expression of genes involved in neurotransmission, such as synapsin-2, GAP43, Vis1, and 143G (Lu et al., 2000), as is also true of the DVD-deficient rat model. ApoE, which is low in the DVD-deficient brain, was also identified as a schizophrenia-susceptibility locus in linkage and association studies (Harrington et al., 1995).

The adult offspring of DVD-deficient dams display a range of behavioral abnormalities, including hyperlocomotion, enhanced sensitivity to psychomimetic drugs, and impaired latent inhibition. Although the mechanisms mediating such behaviors are not clear, the altered behavioral phenotype demonstrates that the effects of prenatal vitamin D deficiency on brain development can persist into adulthood even after the vitamin D is reintroduced at birth.

Rats exposed to DVD deficiency had elevated levels of locomotion across a range of tests, including the hole board and elevated plus maze (Burne et al., 2004) and in the open field (Kesby et al., 2006); this has been referred to as spontaneous hyperlocomotion. This subtle increase in locomotion appears to be a transient response to a novel environment and is sensitive to handling procedures such as restraint (Burne et al., 2006; O’Loan et al., 2007) or restraint and injection (Kesby et al., 2006). One explanation for this phenomenon is an enhanced stress-induced activation of the hypothalamic pituitary axis in DVD-deficient rats. However, we have measured plasma corticosterone levels during and after 30 minutes of restraint stress and found no differences between control and DVD-deficient male rats (Eyles et al., 2006).

With respect to learning and memory, DVD-deficient rats have impaired latent inhibition (Becker et al., 2005). Latent inhibition is a learning phenomenon whereby irrelevant sensory information is filtered unconsciously. Latent inhibition reflects the ability to attend selectively to the important information in the environment and ignore the unimportant, a phenomenon related to both learning and attention. Attentional abnormalities are associated with schizophrenia, and acutely psychotic patients show reduced latent inhibition (Feldon, Shofel, & Weiner, 1991; Lubow & Gewirtz, 1995). Animal models with disrupted latent inhibition are thought to reflect the cognitive or attentional deficits associated with schizophrenia (Grecksch et al., 1999; Moser et al., 2000; Weiner, 2003).

In addition to the subtle and discrete behavioral alterations, DVD-deficient rats exhibit sex-specific behavioral changes in response to psychomimetic agents. DVD-deficient male rats display greater locomotor activity than do DVD-deficient female rats and control male rats in response to the noncompetitive N-methyl-D-aspartic acid receptor (NMDA-R) antagonist, MK-801, in the open field test (Kesby et al., 2006). This effect depends on the timing of the DVD deficiency, because rats experiencing the deficiency during late gestation showed this effect, whereas rats experiencing this deficiency only during early gestation did not (O’Loan et al., 2007). Both the spontaneous and the MK-801-induced hyperlocomotion were abolished by haloperidol, a DA receptor 2 (DA2) blocking agent. Psychomimetic-induced hyperlocomotion in rats has been associated with hyperactivity of mesolimbic DA neurons, a factor thought to be related to psychotic symptoms in schizophrenia (Seeman, 1987). The observation that both enhanced MK-801-induced and spontaneous locomotion in DVD-deficient animals are sensitive to the DA2 receptor blocking agent supports the suggestion that central nervous system DA signaling has been developmentally altered.

In contrast to DVD-deficient males and control females, DVD-deficient females displayed increased locomotion in response to amphetamine. Correspondingly, the density of the DA transporter (DAT) was increased by almost 40% in the caudate-putamen, and the selective affinity for DAT ligands was elevated in the Nac (Kesby et al., 2009b). Amphetamine has been used widely in animal models of schizophrenia. This drug increases striatal DA release (Bardo, Bowling, & Pierce, 1990) through multiple actions, most prominently those incorporating DAT function (Sulzer, Maidment, & Rayport, 1993; Wieczorek & Kruk, 1994; Sulzer et al., 1995; Jones, 1998). Amphetamine also induces a behavioral phenotype that can be blunted by D2 receptor blockade (O’Neill & Shaw, 1999). Hence, increased locomotor sensitivity displayed by animals in response to psychomimetic agents such as amphetamine is considered to be analogous to the enhanced subcortical dopaminergic activity observed in schizophrenia patients (Hietala et al., 1995). In schizophrenia patients, amphetamine can increase the psychotic symptoms by enhancing DA signaling. In healthy individuals, amphetamine can produce psychotic symptoms (Angrist & Vankammen, 1984). Furthermore, low doses of amphetamine that fail to induce psychotic symptoms in healthy individuals can enhance the psychotic symptoms seen in schizophrenia patients (Janowsky et al., 1973).

In conclusion, the experimental data have provided robust evidence demonstrating that DVD deficiency during gestation alters the trajectory of brain development and the structure and function of the adult brain. The DVD model appears to be an informative one for schizophrenia from several perspectives (summarized in table 11.1). With respect to brain development, DVD deficiency leads to alterations in brain morphology, mitosis, programmed cell death, and expression of neurotransmitter-related genes and proteins. Most important, DVD deficiency also induces long-lasting alterations in adult brain outcomes, such as ventriculomegaly and misexpression of genes and proteins involved in mitochondrial function and neurotransmission, which have been reported to be disrupted in patients with schizophrenia. With respect to behavior, the DVD model includes the following: (1) hyperlocomotion, a predominant feature of many animal models of schizophrenia; (2) disrupted latent inhibition, an attentional deficit associated with schizophrenia; and (3) gender-specific sensitivity to psychomimetic drugs, such as MK-801 and amphetamine, as well as antipsychotics, such as haloperidol.

Although clinical research remains central to the field of psychiatry, animal models remain the only practical tool for unraveling the biological mechanisms linking early life disruptions to later neuropsychiatric disorders. The developing human brain is not open to ready observation, and experimental manipulations of normal brain development are clearly not ethical. The DVD model summarized in table 11.1 provides a tool with which to explore a candidate risk factor for schizophrenia. In addition, it has revealed previously unsuspected aspects of neurobiology.

With respect to future studies, first, the predictive validity of the DVD model for schizophrenia should be further examined. The positive symptoms of schizophrenia, such as hallucinations and delusions, are more readily recognized in patients and are generally responsive to antipsychotic medication. Reduced motivation, blunted emotions, social avoidance (negative symptoms of schizophrenia), and impaired cognitive function dramatically limit the patient’s social and functional recovery and are less responsive to antipsychotics. The DVD model shows evidence for psychomotor agitation, a positive symptom of schizophrenia: the animals display increased locomotion following stimulation by psychomimetic drugs. The social and cognitive behavior of the DVD rat needs to be investigated to explore the negative and cognitive symptoms of schizophrenia. Assessment of cognitive deficits in patients with schizophrenia can include a large array of potential measures. Similarly, the assessment of attention, learning, and memory in rodent models could follow many different directions. The need to examine cognitive behaviors in animal models of schizophrenia is also timely because the pharmaceutical industry is investing strongly in the development of products that are designed primarily to ameliorate cognitive deficits rather than to reduce more traditional clinical “targets” such as hallucinations and delusions. Recently, U.S.-based researchers from the Food and Drug Administration, the National Institute of Mental Health, academia, and the pharmaceutical industry published consensus guidelines for clinical trials aimed at the treatment of cognitive symptoms of schizophrenia (Measurement and Treatment Research to Improve Cognition in Schizophrenia, or MATRICS) (Buchanan et al., 2005). The MATRICS panel has grouped the cognitive deficits associated with schizophrenia according to eight “core features” or domains (Buchanan et al., 2005). These include speed of processing, attention, vigilance, working memory, verbal learning, visual learning, reasoning and problem solving, and social cognition (Green et al., 2005). The interdisciplinary panel noted that strong animal models were currently available for working memory, attention, vigilance, and speed of processing.

Secondly, future work is required to clarify the mechanism of action linking hypovitaminosis D and the observed brain changes. In addition, it will be important to clarify how these changes are associated with altered behaviors. Finally, although the results from the DVD-deficient animal experiments indicated that brain structure and function are altered in rodents, it remains to be seen if maternal vitamin D deficiency is directly associated with schizophrenia in humans. Schizophrenia is associated with a substantial burden of disability. In the absence of major advances in the efficacy of treatments, interventions that offer the prospect of reducing the incidence of the disorder should be pursued vigorously. If future studies confirm the association between DVD deficiency and risk of schizophrenia, then it raises the tantalizing prospect of primary prevention, in a manner comparable to folate supplementation and the prevention of spina bifida.

We are grateful for the support of the National Health and Medical Research Council. James Kesby kindly provided figure 11.2.

Almeras, L., Eyles, D., Benech, P., Laffite, D., Villard, C., Patatian, A., Boucraut, J., Mackay-Sim, A., McGrath, J., & Feron, F. (2007). Developmental vitamin D deficiency alters brain protein expression in the adult rat: Implications for neuropsychiatric disorders. Proteomics 7(5): 769–780.

Angrist, B. & Vankammen, D. P. (1984). CNS stimulants as tools in the study of schizophrenia. Trends in Neuroscience 7(10): 388–390.

Balabanova, S., Richter, H. P., Antoniadis, G., Homoki, J., Kremmer, N., Hanle, J., & Teller, W. M. (1984). 25-Hydroxyvitamin D, 24, 25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human cerebrospinal fluid. Klinische Wochenschrift 62(22): 1086–1090.

Bardo, M. T., Bowling, S. L., & Pierce, R. C. (1990). Changes in locomotion and dopamine neurotransmission following amphetamine, haloperidol, and exposure to novel environmental stimuli. Psychopharmacology 101(3): 338–343.

Becker, A., Eyles, D. W., McGrath, J. J., & Grecksch, G. (2005). Transient prenatal vitamin D deficiency is associated with subtle alterations in learning and memory functions in adult rats. Behavioural Brain Research 161(2): 306–312.

Bouillon, R., Verstuyf, A., Branisteanu, D., Waer, M., & Mathieu, C. (1995). Immune modulation by vitamin D analogs in the prevention of autoimmune diseases. Koninklijke Academie voor Geneeskunde van België 57(5): 371–385; discussion 385–387.

Bradbury, T. N. & Miller, G. A. (1985). Season of birth in schizophrenia: A review of evidence, methodology, and etiology. Psychological Bulletin 98(3): 569–594.

Brown, J., Bianco, J. I., McGrath, J. J., & Eyles, D. W. (2003). 1,25-dihydroxyvitamin D3 induces nerve growth factor, promotes neurite outgrowth and inhibits mitosis in embryonic rat hippocampal neurons. Neuroscience Letters 343(2): 139–143.

Buchanan, R. W., Davis, M., Goff, D., Green, M. F., Keefe, R. S., Leon, A. C., Nuechterlein, K. H., Laughren, T., Levin, R., Stover, E., et al. (2005). A summary of the FDA-NIMH-MATRICS workshop on clinical trial design for neurocognitive drugs for schizophrenia. Schizophrenia Bulletin 31(1): 5–19.

Burkert, R., McGrath, J., & Eyles, D. (2003). Vitamin D receptor expression in the embryonic brain. Neuroscience Research Communications 33(2): 67–71.

Burne, T. H., Becker, A., Brown, J., Eyles, D. W., Mackay-Sim, A., & McGrath, J. J. (2004). Transient prenatal vitamin D deficiency is associated with hyperlocomotion in adult rats. Behavioural Brain Research 154(2): 549–555.

Burne, T. H., O’Loan, J., McGrath, J. J., & Eyles, D. W. (2006). Hyperlocomotion associated with transient prenatal vitamin D deficiency is ameliorated by acute restraint. Behavioural Brain Research 174(1): 119–124.

Cantor-Graae, E., Zolkowska, K., & McNeil, T. F. (2005). Increased risk of psychotic disorder among immigrants in Malmo: A 3-year first-contact study. Psychological Medicine 35(8): 1155–1163.

Christakos, S., Dhawan, P., Peng, X., Obukhov, A. G., Nowycky, M. C., Benn, B. S., Zhong, Y., Liu, Y., & Shen, Q. (2007). New insights into the function and regulation of vitamin D target proteins. Journal of Steroid Biochemistry and Molecular Biology 103(3–5): 405–410.

Cui, X., McGrath, J. J., Burne, T. H., Mackay-Sim, A., & Eyles, D. W. (2007). Maternal vitamin D depletion alters neurogenesis in the developing rat brain. International Journal of Developmental Neuroscience 25(4): 227–232.

Davies, G., Welham, J., Chant, D., Torrey, E. F., & McGrath, J. (2003). A systematic review and meta-analysis of Northern Hemisphere season of birth studies in schizophrenia. Schizophrenia Bulletin 29(3): 587–593.

Deluca, H. F. & Cantorna, M. T. (2001). Vitamin D: Its role and uses in immunology. FASEB Journal 15(14): 2579–2585.

Ebert, R., Schutze, N., Adamski, J., & Jakob, F. (2006). Vitamin D signaling is modulated on multiple levels in health and disease. Molecular and Cellular Endocrinology 248(1–2): 149–159.

Eyles, D., Almeras, L., Benech, P., Patatian, A., Mackay-Sim, A., McGrath, J., & Feron, F. (2007). Developmental vitamin D deficiency alters the expression of genes encoding mitochondrial, cytoskeletal and synaptic proteins in the adult rat brain. Journal of Steroid Biochemistry and Molecular Biology 103(3–5): 538–545.

Eyles, D., Anderson, C., Ko, P., Jones, A., Thomas, A., Burne, T., Mortensen, P. B., Norgaard-Pedersen, B., Hougaard, D. M., & McGrath, J. (2009). A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots. Clinica Chimica Acta 403(1–2): 145–151.

Eyles, D., Brown, J., Mackay-Sim, A., McGrath, J., & Feron, F. (2003). Vitamin D3 and brain development. Neuroscience 118(3): 641–653.

Eyles, D. W., Rogers, F., Buller, K., McGrath, J. J., Ko, P., French, K., & Burne, T. H. (2006). Developmental vitamin D (DVD) deficiency in the rat alters adult behaviour independently of HPA function. Psychoneuroendocrinology 31(8): 958–964.

Eyles, D. W., Smith, S., Kinobe, R., Hewison, M., & McGrath, J. J. (2005). Distribution of the vitamin D receptor and 1 alpha-hydroxylase in human brain. Journal of Chemical Neuroanatomy 29(1): 21–30.

Feldon, J., Shofel, A., & Weiner, I. (1991). Latent inhibition is unaffected by direct dopamine agonists. Pharmacology Biochemistry and Behavior 38(2): 309–314.

Feron, F., Burne, T. H., Brown, J., Smith, E., McGrath, J. J., Mackay-Sim, A., & Eyles, D. W. (2005). Developmental vitamin D3 deficiency alters the adult rat brain. Brain Research Bulletin 65(2): 141–148.

Fu, G. K., Lin, D., Zhang, M. Y., Bikle, D. D., Shackleton, C. H., Miller, W. L., & Portale, A. A. (1997). Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Molecular Endocrinology 11(13): 1961–1970.

Fujimoto, T., Nakano, T., Takano, T., Hokazono, Y., Asakura, T., & Tsuji, T. (1992). Study of chronic schizophrenics using 31P magnetic resonance chemical shift imaging. Acta Psychiatrica Scandinavica 86(6): 455–462.

Garcion, E., Wion-Barbot, N., Montero-Menei, C. N., Berger, F., & Wion, D. (2002). New clues about vitamin D functions in the nervous system. Trends in Endocrinology and Metabolism 13(3): 100–105.

Gascon-Barre, M. & Huet, P. M. (1983). Apparent [3H]1,25-dihydroxyvitamin D3 uptake by canine and rodent brain. American Journal of Physiology 244(3): E266–E271.

Grecksch, G., Bernstein, H. G., Becker, A., Hollt, V., & Bogerts, B. (1999). Disruption of latent inhibition in rats with postnatal hippocampal lesions. Neuropsychopharmacology 20(6): 525–532.

Green, M. F., Olivier, B., Crawley, J. N., Penn, D. L., & Silverstein, S. (2005). Social cognition in schizophrenia: Recommendations from the measurement and treatment research to improve cognition in schizophrenia new approaches conference. Schizophrenia Bulletin 31(4): 882–887.

Hager, G., Formanek, M., Gedlicka, C., Thurnher, D., Knerer, B., & Kornfehl, J. (2001). 1,25(OH)2 vitamin D3 induces elevated expression of the cell cycle-regulating genes P21 and P27 in squamous carcinoma cell lines of the head and neck. Acta Oto-Laryngologica 121(1): 103–109.

Harrington, C. R., Roth, M., Xuereb, J. H., McKenna, P. J., & Wischik, C. M. (1995). Apolipoprotein E type epsilon 4 allele frequency is increased in patients with schizophrenia. Neuroscience Letters 202(1–2): 101–104.

Harrison, L. E., Wang, Q. M., & Studzinski, G. P. (1999). 1,25-dihydroxyvitamin D(3)-induced retardation of the G(2)/M traverse is associated with decreased levels of p34(cdc2) in HL60 cells. Journal of Cellular Biochemistry 75(2): 226–234.

Heaney, R. P. (2007). Vitamin D endocrine physiology. Journal of Bone and Mineral Research 22: V25–V27.

Hietala, J., Syvalahti, E., Vuorio, K., Rakkolainen, V., Bergman, J., Haaparanta, M., Solin, O., Kuoppamaki, M., Kirvela, O., Ruotsalainen, U., et al. (1995). Presynaptic dopamine function in striatum of neuroleptic-naive schizophrenic patients. Lancet 346(8983): 1130–1131.

Holick, M. F. (1988). Skin: Site of the synthesis of vitamin-D and a target tissue for the active form, 1,25-Dihydroxyvitamin-D3. Annals of the New York Academy of Sciences 548: 14–26.

Holick, M. F. (2007). Vitamin D deficiency. New England Journal of Medicine 357(3): 266–281.

Holick, M. F., Matsuoka, L. Y., & Wortsman, J. (1995). Regular use of sunscreen on vitamin D levels. Archives of Dermatology 131(11): 1337–1339.

Janowsky, D. S., el-Yousel, M. K., Davis, J. M., & Sekerke, H. J. (1973). Provocation of schizophrenic symptoms by intravenous administration of methylphenidate. Archives of General Psychiatry 28(2): 185–191.

Jones, S. R., Gainetdinov, R. R., Wightman, R. M., & Caron, M. G. (1998). Mechanisms of amphetamine action revealed in mice lacking the dopamine transporter. Journal of Neuroscience 18(6): 1979–1986.

Kesby, J. P., Burne, T. H., McGrath, J. J., & Eyles, D. W. (2006). Developmental vitamin D deficiency alters MK 801-induced hyperlocomotion in the adult rat: An animal model of schizophrenia. Biological Psychiatry 60(6): 591–596.

Kesby, J. P., Cui, X., Ko, P., McGrath, J. J., Burne, T. H., & Eyles, D. W. (2009a). Developmental vitamin D deficiency alters dopamine turnover in neonatal rat forebrain. Neuroscience Letters 461(2): 155–158.

Kesby, J. P., Cui, X., O’Loan, J., McGrath, J., Burne, T. H. J., & Eyles, D. W. (2009b). Developmental vitamin D deficiency alters dopamine-mediated behaviors and dopamine transporter function in adult female rats. Psychopharmacology 208(1): 159–168.

Ko, P., Burkert, R., McGrath, J., & Eyles, D. (2004). Maternal vitamin D3 deprivation and the regulation of apoptosis and cell cycle during rat brain development. Brain Research: Developmental Brain Research 153(1): 61–68.

Lauer, M., Senitz, D., & Beckmann, H. (2001). Increased volume of the nucleus accumbens in schizophrenia. Journal of Neural Transmission 108(6): 645–660.

Lin, R. & White, J. H. (2004). The pleiotropic actions of vitamin D. Bioessays 26(1): 21–28.

Liu, M., Lee, M. H., Cohen, M., Bommakanti, M., & Freedman, L. P. (1996). Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937. Genes and Development 10(2): 142–153.

Lu, F., Selak, M., O’Connor, J., Croul, S., Lorenzana, C., Butunoi, C., & Kalman, B. (2000). Oxidative damage to mitochondrial DNA and activity of mitochondrial enzymes in chronic active lesions of multiple sclerosis. Journal of the Neurological Sciences 177(2): 95–103.

Lubow, R. E. & Gewirtz, J. C. (1995). Latent inhibition in humans: Data, theory, and implications for schizophrenia. Psychological Bulletin 117(1): 87–103.

Marcelis, M., Navarro-Mateu, F., Murray, R., Selten, J. P., & van Os, J. (1998). Urbanization and psychosis: A study of 1942–1978 birth cohorts in The Netherlands. Psychological Medicine 28(4): 871–879.

McCann, J. C. & Ames, B. N. (2008). Is there convincing biological or behavioral evidence linking vitamin D deficiency to brain dysfunction? FASEB Journal 22(4): 982–1001.

McGrath, J., Eyles, D., Mowry, B., Yolken, R., & Buka, S. (2003). Low maternal vitamin D as a risk factor for schizophrenia: A pilot study using banked sera. Schizophrenia Research 63(1–2): 73–78.

McGrath, J., Feron, F., & Eyles, D. (2001). Vitamin D: The neglected neurosteroid? Trends in Neuroscience 24(10): 570–572.

McGrath, J., Iwazaki, T., Eyles, D., Burne, T., Cui, X., Ko, P., & Matsumoto, I. (2008). Protein expression in the nucleus accumbens of rats exposed to developmental vitamin D deficiency. PLoS One 3(6): e2383.

McGrath, J., Saari, K., Hakko, H., Jokelainen, J., Jones, P., Järvelin, M. R., Chant, D., & Isohanni, M. (2004). Vitamin D supplementation during the first year of life and risk of schizophrenia: A Finnish birth-cohort study. Schizophrenia Research 67(2–3): 237–245.

McGrath, J. J., Kimlin, M. G., Saha, S., Eyles, D. W., & Parisi, A. V. (2001). Vitamin D insufficiency in south-east Queensland. Medical Journal of Australia 174(3): 150–151.

Mortensen, P. B., Pedersen, C. B., Westergaard, T., Wohlfahrt, J., Ewald, H., Mors, O., Andersen, P. K., & Melbye, M. (1999). Effects of family history and place and season of birth on the risk of schizophrenia. New England Journal of Medicine 340(8): 603–608.

Moser, P. C., Hitchcock, J. M., Lister, S., & Moran, P. M. (2000). The pharmacology of latent inhibition as an animal model of schizophrenia. Brain Research Reviews 33(2–3): 275–307.

Nagpal, S., Na, S., & Rathnachalam, R. (2005). Noncalcemic actions of vitamin D receptor ligands. Endocrine Reviews 26(5): 662–687.

Neveu, I., Naveilhan, P., Menaa, C., Wion, D., Brachet, P., & Garabedian, M. (1994). Synthesis of 1,25-dihydroxyvitamin D3 by rat brain macrophages in vitro. Journal of Neuroscience Research 38(2): 214–220.

Nguyen, L., Rigo, J. M., Rocher, V., Belachew, S., Malgrange, B., Rogister, B., Leprince, P., & Moonen, G. (2001). Neurotransmitters as early signals for central nervous system development. Cell Tissue Research 305(2): 187–202.

O’Loan, J., Eyles, D. W., Kesby, J., Ko, P., McGrath, J. J., & Burne, T. H. (2007). Vitamin D deficiency during various stages of pregnancy in the rat: Its impact on development and behaviour in adult offspring. Psychoneuroendocrinology 32(3): 227–234.

O’Neill, M. F. & Shaw, G. (1999). Comparison of dopamine receptor antagonists on hyperlocomotion induced by cocaine, amphetamine, MK-801 and the dopamine D-1 agonist C-APB in mice. Psychopharmacology 145(3): 237–250.

Pardridge, W. M., Sakiyama, R., & Coty, W. A. (1985). Restricted transport of vitamin D and A derivatives through the rat blood-brain barrier. Journal of Neurochemistry 44(4): 1138–1141.

Pedersen, C. B. & Mortensen, P. B. (2001). Family history, place and season of birth as risk factors for schizophrenia in Denmark: A replication and reanalysis. British Journal of Psychiatry 179(1): 46–52.

Polly, P., Danielsson, C., Schrader, M., & Carlberg, C. (2000). Cyclin C is a primary 1alpha,25-dihydroxyvitamin D(3) responding gene. Journal of Cellular Biochemistry 77(1): 75–81.

Raiten, D. J. & Picciano, M. F. (2004). Vitamin D and health in the 21st century: Bone and beyond. Executive summary. American Journal of Clinical Nutrition 80(6 Suppl): 1673S–1677S.

Reichrath, J., Lehmann, B., Carlberg, C., Varani, J., & Zouboulis, C. C. (2007). Vitamins as hormones. Hormone and Metabolic Research 39(2): 71–84.

Ruediger, T. & Bolz, J. (2007). Neurotransmitters and the development of neuronal circuits. Advances in Experimental Medicine and Biology 621: 104–115.

Saha, S., Chant, D. C., Welham, J. L., & McGrath, J. J. (2006). The incidence and prevalence of schizophrenia varies with latitude. Acta Psychiatrica Scandinavica 114(1): 36–39.

Seeman, P. (1987). Dopamine receptors and the dopamine hypothesis of schizophrenia. Synapse 1(2): 133–152.

Sulzer, D., Chen, T. K., Lau, Y. Y., Kristensen, H., Rayport, S., & Ewing, A. (1995). Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. Journal of Neuroscience 15(5): 4102–4108.

Sulzer, D., Maidment, N. T., & Rayport, S. (1993). Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons. Journal of Neurochemistry 60(2): 527–535.

Sutherland, M. K., Somerville, M. J., Yoong, L. K., Bergeron, C., Haussler, M. R., & McLachlan, D. R. (1992). Reduction of vitamin D hormone receptor mRNA levels in Alzheimer as

compared to Huntington hippocampus: Correlation with calbindin-28k mRNA levels. Brain Research: Molecular Brain Research 13(3): 239–250.

Torrey, E., Miller, J., Rawlings, R., & Yolken, R. (1997). Seasonality of births in schizophrenia and bipolar disorder: A review of the literature. Schizophrenia Research 28(1): 1–38.

Torrey, E. F. & Miller, J. (1997). Season of birth and schizophrenia: Southern hemisphere data. Australian and New Zealand Journal of Psychiatry 31(2): 308–309.

Veenstra, T. D., Prufer, K., Koenigsberger, C., Brimijoin, S. W., Grande, J. P., & Kumar, R. (1998). 1,25-Dihydroxyvitamin D3 receptors in the central nervous system of the rat embryo. Brain Research 804(2): 193–205.

Volz, H. R., Riehemann, S., Maurer, I., Smesny, S., Sommer, M., Rzanny, R., Holstein, W., Czekalla, J., & Sauer, H. (2000). Reduced phosphodiesters and high-energy phosphates in the frontal lobe of schizophrenic patients: A (31)P chemical shift spectroscopic-imaging study. Biological Psychiatry 47(11): 954–961.

Weiner, I. (2003). The “two-headed” latent inhibition model of schizophrenia: Modeling positive and negative symptoms and their treatment. Psychopharmacology (Berl) 169(3–4): 257–297.

Westerink, B. H. C. (1985). Sequence and significance of dopamine metabolism in the rat brain. Neurochemistry International 7(2): 221–227.

Wieczorek, W. J. & Kruk, Z. L. (1994). Differential action of (+)-Amphetamine on electrically-evoked dopamine overflow in rat-brain slices containing corpus striatum and nucleus-accumbens. British Journal of Pharmacology 111(3): 829–836.

Wood, P. L., Kim, H. S., & Marien, M. R. (1987). Intracerebral dialysis: Direct evidence for the utility of 3-MT measurements as an index of dopamine release. Life Sciences 41(1): 1–5.

Zehnder, D., Bland, R., Williams, M. C., McNinch, R. W., Howie, A. J., Stewart, P. M., & Hewison, M. (2001). Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase. Journal of Clinical Endocrinology and Metabolism 86(2): 888–894.

Modality	DVD-Deficient Phenotype
Brain development	Enlarged lateral ventricles (Eyles et al., 2003) Increase mitosis and reduced programmed cell death (Eyles et al., 2003; Ko, McGrath, & Eyles, 2004) Increased neurosphere formation (Cui et al., 2007) Disrupted dopamine turnover (Kesby et al., 2009a)
Adult brain	Enlarged lateral ventricles (Feron et al., 2005) Mis-expression of genes and proteins involved in mitochondria function and neurotransmission (Almeras et al., 2007; McGrath et al., 2008; Kesby et al., 2009a) Increased DAT density and affinity in mesolimbic system (Kesby et al., 2009b)
Adult behavior	Hyperlocomotion (Burne et al., 2006; Kesby et al., 2006; O’Loan et al., 2007) Disrupted latent inhibition (LI) (Becker et al., 2005) Hyperlocomotion in response to MK-801(Kesby et al., 2006; O’Loan et al., 2007) Hyperlocomotion in response to amphetamine (Kesby et al., 2009b) Sensitivity to haloperidol (Kesby et al., 2006)