WO2003063585A1

WO2003063585A1 - Animal model

Info

Publication number: WO2003063585A1
Application number: PCT/AU2003/000102
Authority: WO
Inventors: Alan Mackay-Sim; Francois Pierre Feron; John Joseph Mcgrath; Thomas Henry Johnston Burne; Darryl Walter Eyles
Original assignee: Griffith University; The State Of Queensland Acting Through Its Department Of Health
Priority date: 2002-01-31
Filing date: 2003-01-31
Publication date: 2003-08-07

Abstract

The invention relates to a method for modifying a behaviour and/or morphology of an animal, an animal having modified behaviour and/or morphology and uses of the animal. The animal is a progeny of a mother having at least a reduced amount of vitamin D prior to giving birth to the animal. The animal may also have at least a reduced amount of vitamin D after birth. The modified behaviour and/or morphology may correlate with a known disorder, such as a neuro-psychiatric disorder, for example schizophrenia. The animal may be useful as an animal model for a disorder and may be used for identifying a behaviour modifying molecule.

Description

TITLE "ANIMAL MODEL" FIELD OF THE INVENTION THIS INVENTION relates to a method for modifying a behaviour and/or morphology of an animal, an animal having a modified behaviour and/or morphology and uses of the animal. The modified behaviour and/or morphology may correlate with a known disorder. The change in behaviour may be associated with a neuro-psychiatric disorder, for example schizophrenia. The animal may be useful as an animal model for a disorder and may be used for screening an active molecule such as a drug for treating a disorder.

BACKGROUND OF THE INVENTION

Animal models are useful when studying a disorder in humans.

Experiments not permissible for humans may be performed on the animal model to better understand the disorder and to explore potential treatments therefor. The animal model typically presents with a phenotype at least partially consistent with a disorder to be studied.

Neuro-psychiatric disorders include, for example, schizophrenia, multiple sclerosis, anxiety, autism, Alzheimer's disease, Parkinson's disease, depression, mania, attention deficit/hyperactivity disorder and dementia. The disorder may be a disorder characterized by increased dopaminergic tone, for example disorders associated with hyperlocomotion. Schizophrenia may be such a disorder as described by Mohn et al, 1999, Cell 98427. People presenting with such disorders often are prescribed medication to provide some relief from behavioral changes associated with the disorder. Although there are some medications currently available, development of new medications is desirable to improve treatment and patient comfort in relation to these disorders.

Schizophrenia is a group of brain disorders with great variability in neurobiological correlates, symptom profile, course of illness and response to treatment. It is generally accepted that a mix of genetic and environmental risk factors are involved in increasing susceptibility to schizophrenia, however, aetiology of schizophrenia is still poorly understood.

One hypothesis, "neurodevelopmental hypothesis", proposes that schizophrenia is related to abnormal brain development [Weinberger, 1986; Nasrallah, 1990; Jones, 1991]. Evidence supporting this has been reviewed (eg. [McGrath, 1995; Raedler, 1998; Harrison, 1997]). While the neurodevelopmental hypothesis of schizophrenia provides a model with respect to timing of a "lesion", it does not advance any specific cellular mechanisms or genetic pathways that could explain the pathogenesis ofthe disorder.

Family, adoption and twin studies have provided compelling evidence that genetic factors influence the risk of developing schizophrenia [Mowry, 1997]. Less widely appreciated is that these same studies also provide powerful indirect evidence for a role of non-genetic risk factors for schizophrenia [Reiss, 1991]. However, a search for non-genetic risk factors for schizophrenia has not been extensively conducted and accordingly, non- genetic risk factors may be under considered.

To study and test hypothesis in relation to causes and treatments for neuro-psychiatric disorders such as schizophrenia, it is useful to perform experiments on an animal model. A method of making a animal model of schizophrenia using chemical treatment or surgical alterations has been described by Swerdlow, 1994. Methods requiring chemical and surgical treatments, however, often disrupt one or more neurotransmitter systems. Such treatment to produce an animal model of schizophrenia may obscure interpretation of effectiveness of a tested drug. For example, typically a drug used to treat schizophrenia also alters one or more neurotransmitter systems, such as dopamine, serotonin, glutamate and gamma amino butyric acid systems. Testing such a drug using an animal model whereby the animal is made by directly disrupting a neurotransmitter system may be considered circular, ie. a schizophrenic animal is made by altering a neurotransmitter system and then the animal is treated with a drug that affects a neurotransmitter system.

US Patent No. 5,720,936 relates to a transgenic animal for assaying compounds for treating Alzheimer's disease. A transgenic animal may be useful to express a desired gene or to knock-out or mutate an endogenous gene thereby resulting in a disease phenotype. However, producing a transgenic animal is typically expensive and time consuming, and requires identification and isolation of a gene involved with a particular disease. Identification and isolation of such a gene is often difficult and time consuming and may be further complicated if a disease is caused by multiple gene products.

US Patent No. 5,549,884 relates to a schizophrenic animal model wherein the animal is brain damaged while prepubescent. The brain damage consists of a ventral hippocampus lesion induced by exposure of the hippocampus region to a neurotoxin. This animal model requires physical damage to the animal's brain that is time consuming and may also result in damage not associated with schizophrenia. It has been proposed that low prenatal 1 ,25-dihydroxyvitamin

D₃ (calcitriol) may increase a risk for schizophrenia in a newborn human [McGrath, 1999], incorporated herein by reference.

SUMMARY OF THE INVENTION The inventors have realized a need for a non-transgenic non- human animal model for a neuro-psychiatric disorder that does not require chemical and/or surgical alteration.

The inventors have surprisingly developed a non-human animal model that may be useful in screening drugs for treatment of neuro- psychiatric disorders such as schizophrenia. An animal model made according to the method of the invention has advantages of not requiring the animal's brain to be surgically damaged, there is no requirement to identify a specific gene to knock-out or mutate and there is no direct interference with a neurotransmitter system by a chemical agent.

In a first aspect, the invention provides a method for producing a non-human animal characterized by a modified behavior when compared with a normal animal, the method including the step of at least reducing an amount of vitamin D, vitamin D precursor or vitamin D-like compound of the mother of the animal.

Preferably, the amount of vitamin D, vitamin D precursor or vitamin D-like compound is reduced in the mother at least prior to weaning of the animal. The amount of vitamin D, vitamin D precursor or vitamin D-like compound may be reduced in the mother prior to birth of the animal.

The amount of vitamin D, vitamin D precursor or vitamin D-like compound may be reduced in the mother prior to pregnancy.

Preferably, the amount of vitamin D, vitamin D precursor or vitamin D-like compound is reduced in the mother at least two weeks prior to pregnancy.

More preferably, the amount of vitamin D, vitamin D precursor or vitamin D-like compound is reduced in the mother at least four weeks prior to pregnancy. The amount of vitamin D, vitamin D precursor or vitamin D-like compound of the mother may be measured as a 25 Hydroxy vitamin D plasma concentration of less than 15 ng/ml for a rat.

Preferably, the amount of vitamin D, vitamin D precursor or vitamin D-like compound ofthe mother is measured as a 25 Hydroxy vitamin D plasma concentration of less than 5 ng/ml for a rat.

The amount of vitamin D, vitamin D precursor or vitamin D-like compound of the mother is preferably reduced by feeding said mother a diet deficient in an amount of vitamin D, vitamin D precursor or vitamin D-like compound.

The animal born from the mother preferably has at least a reduced amount of vitamin D, vitamin D precursor or vitamin D-like compound. Preferably, the amount of vitamin D, vitamin D precursor or vitamin D-like compound of the animal born from the mother is reduced at least during early development.

The amount of vitamin D, vitamin D precursor or vitamin D-like compound ofthe animal born from the mother may be reduced at least from birth to weaning.

The amount of vitamin D, vitamin D precursor or vitamin D-like compound ofthe animal born from the mother may be reduced at least from birth to adulthood.

The amount of vitamin D, vitamin D precursor or vitamin D-like compound of the animal born from the mother is preferably measured as a plasma concentration of 25 Hydroxy vitamin D less than about 30 ng/ml at least for a rat.

Preferably, the plasma concentration of 25 Hydroxy vitamin D is less than about 5 ng/ml at least for a rat.

The amount of vitamin D, vitamin D precursor or vitamin D-like compound of the animal born from the mother is preferably reduced by feeding the animal a diet comprising a deficient amount of vitamin D, vitamin D precursor or vitamin D-like compound.

The diet may comprise a reduced amount or no detectable amount of vitamin D, vitamin D precursor or vitamin D-like compound.

The modified behavior of the animal may include hyperlocomotion, decreased social interaction and/or a change in prepulse inhibition.

The animal born from the mother may be used as a means for assessing a behavioral disorder.

Preferably, the behavioral disorder is a neuro-psychiatric disorder. More preferably, the neuro-psychiatric disorder is characterized by hyperlocomotion, decreased social interaction and/or a change in prepulse inhibition when compared with a normal animal.

Preferably, the neuro-psychiatric disorder is selected from the group consisting of: schizophrenia, multiple sclerosis, anxiety, autism, Alzheimer's disease, Parkinson's disease, depression, mania, attention deficit/hyperactivity disorder and dementia.

Preferably, the neuro-psychiatric disorder is characterised by increased dopaminergic tone. Most preferably, the neuro-psychiatric disorder is schizophrenia.

The animal may further be characterized by a modified morphology when compared with a normal animal. The mother and animal born from the mother are preferably not exposed to ultra violet B (UVB).

Preferably, the mother and animal born from the mother are only exposed to incandescent light.

Preferably, the animal is a mammal. More preferably, the mammal is a rat or mouse.

In a second aspect, the invention provides a non-human animal produced according to the method of the first aspect.

In a third aspect, the invention provides a non-human animal characterized by a modified behavior when compared with a normal non- human animal, said non-human animal being a progeny of a mother having at least a reduced amount of vitamin D, vitamin D precursor or vitamin D-like compound at least prior to giving birth to said non-human animal.

The non-human animal may have at least a reduced amount of vitamin D, vitamin D precursor or vitamin D-like compound. The reduced amount of vitamin D, vitamin D precursor or vitamin D-like compound is preferably a result of a diet deficient in vitamin D, vitamin D precursor or vitamin D-like compound.

The modified behavior may be characteristic of a disorder. The disorder is preferably a neuro-psychiatric disorder.

The disorder is preferably selected from the group consisting of: schizophrenia, multiple sclerosis, anxiety, autism, Alzheimer's disease, Parkinson's disease, depression, mania, attention deficit/hyperactivity disorder and dementia.

The neuro-psychiatric disorder may be characterised by increased dopaminergic tone.

The neuro-psychiatric disorder is preferably schizophrenia.

The non-human animal is preferably a mammal. The mammal is preferably a rat or mouse.

In a fourth aspect, the invention provides a method for identifying a behavior modifying molecule including the steps of:

(i) administering at least one candidate molecule to an animal of the second or third aspect; and (ii) assessing the animal for a behavioral change in response to said candidate molecule(s) to determine if there is a change from the modified behavior, wherein a change from the modified behavior indicates said candidate molecule(s) is a behavior modifying molecule. Preferably, the modified behavior is selected from the group consisting of: hyperlocomotion, decreased social interaction and a change in pre-pulse inhibition when compared with a normal animal.

Preferably, the modified behaviour is characteristic of a behavioural disorder.

The behaviour disorder is preferably a neuro-psychiatric disorder.

The neuro-psychiatric disorder may be characterized by increased dopaminergic tone.

Preferably, the neuro-psychiatric disorder is selected from the group consisting of: schizophrenia, multiple sclerosis, anxiety, autism, Alzheimer's disease, Parkinson's disease, depression, mania, attention deficit/hyperactivity disorder and dementia. The neuro-psychiatric disorder is preferably schizophrenia.

The behavior modifying molecule may be a known drug for treating a disorder other than that identified by the method of the fourth aspect.

The behavior modifying molecule may be a known drug for treating a disorder other than a neuro-psychiatric disorder.

The behavior modifying molecule may be a known drug for treating a non-schizophrenic disorder.

The behavior modifying molecule may be a novel drug.

Preferably, the animal is at least 10 weeks old before being administered with a candidate molecule.

It will be appreciated by one skilled in the art that the animal described herein characterized by a modified behavior, e.g. an abnormal behavior, may be useful as an animal model for any suitable disorder wherein the behavior of the animal is at least partially consistent with, or characteristic of, behavior presented by the disorder. The animal may be useful as an animal model for a neuro-psychiatric disorder, including for example schizophrenia, multiple sclerosis, anxiety, autism, Alzheimer's disease, Parkinson's disease, depression, mania, attention deficit/hyperactivity disorder, dementia and neuro-degenerative disorders in general. The animal model described herein is particularly useful as a model for schizophrenia.

Throughout this specification unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion ofthe stated integers or group of integers or steps but not the exclusion of any other integer or group of integers.

DESCRIPTION OF THE FIGURES AND TABLES In order that the invention may be readily understood and put into practical effect, preferred embodiments will now be described by way of example with reference to the accompanying figures and tables, wherein:

FIG. 1 is a graph showing prepulse inhibition is reduced in chronic vitamin D depleted adults. Bars show cumulative prepulse inhibition (percent inhibition in prepulse trials compared to test pulse alone trials) for three prepulse inhibition trials (5dB, 10dB, 15dB; dB=decibels) for males (A, C) and females (B,D) tested at 5 weeks (A, B) and 10 weeks (C, D). Note that the Late group (depleted of vitamin D from weeks 5-10) were only tested at 10 weeks.

FIG. 2 shows graphs of amplitudes ofthe startle responses to 120 dB stimuli at 5 Weeks (A) and 10 Weeks (B) normalized by body weight. At 10 weeks Deplete All males had larger startle responses at 10 Weeks when compared to body weight.

FIG. 3 shows graphs of response of an animal to auditory stimulus (dB). Auditory sensitivity is not affected by vitamin D status.

Amplitude of startle response (Y-axis, arbitrary units) versus stimulus intensity (dB) at 5 weeks (A) and 10 weeks (B). The response threshold is similar for all groups at 90dB.

FIG.4 shows graphs illustrating that startle response relative to body weight is increased in chronic vitamin D depleted adults. The bars show cumulative startle response magnitude for the three loudest stimuli (100dB,

110dB, 120dB) for males (A, C) and females (B,D) tested at 5 weeks (A, B) and 10 weeks (C, D).

FIG. 5 shows graphs illustrating that startle response latency is increased in chronic vitamin D depleted adults. The bars show cumulative latency for the three loudest stimuli (1 OOdB, 110dB, 120dB) for males (A, C) and females (B,D) tested at 5 weeks (A, B) and 10 weeks (C, D). FIG.6 shows graphs of an open field test. The graphs show the total distance moved in the open field at 5 Weeks (A) and 10 Weeks (B). Note the different Y-axis scales in A and B.

FIG. 7 shows graphs of time structure of locomotion in Open Field test. The graphs show the distance moved (cm) in each 10s time block over the 200s test period. The animals were more active at 5 Weeks (A, B) than at 10 Weeks (C, D) and females (B, D) were more active than males (A, C). The vitamin D depleted adult group was less active than the others at 5 weeks but this was not evident at 10 weeks.

FIG.8(a) shows plasma 25 hydroxy-Vitamin D₃ (250H Vitamin D) concentrations in dams immediately prior to mating and in respective offspring (neonates). In animals kept under normal vitamin D conditions, levels were higher in neonates compared with dams * (P<0.001, n =14). FIG. 8(b) shows serum calcium levels were greater in the deplete neonates * (P<0.05, n=8). Bar graphs indicate means + SEM.

FIG. 9 shows plasma 25 hydroxy-Vitamin D₃ (250H Vitamin D) concentrations in dams immediately prior to mating and in respective neonates. Neonate values are shown respectively for males (M) and females (F). This graph illustrates sex did not have an effect in the neonates (P> 0.05), 250H Vitamin D levels were greater in normal neonates when compared with mothers (by simple ANOVA), and 250H Vitamin D levels were lower in depleted dams and male and female neonates when compared with controls and 250H Vitamin D levels in all depleted groups were similar.

FIG. 10 is a micrograph showing ventricular enlargement in the brains of vitamin D deplete neonates. There was no difference between male and female lateral ventricle volumes nor was there any asymmetry. Therefore ventricle volumes (left and right) were combined and animals were assessed independent of sex. The top image is of a normal neonate brain, the bottom image is of a brain of a vitamin D depleted neonate.

FIG. 11(a) shows a graph of lateral ventricular volumes. * (P<0.01 n = 12).

FIG. 11 (b) shows a graph of ventricular volumes as a ratio of hemisphere volume * (P<0.05 n = 12). Bar graphs indicate means ± SEM.

FIG. 12 shows vitamin D depletion did not alter cell densities in any brain region investigated. Bar graphs indicate means ± SEM (n=10). FIG. 13(a) shows vitamin D depletion increased the percentage of cells undergoing mitosis in the dentate * (P<0.001), hypothalamus * (P<0.05) and basal ganglia/amyg * (P<0.05), but not the cingulate.

FIG. 13(b) shows mitotic cells exhibiting characteristic immunohistochemical staining with anti-PCNA antibody. Bar graphs indicate means + SEM (n=10).

FIG. 14 shows vitamin D depletion appeared to decrease the percentage of cells undergoing apoptosis in all four brain regions investigated, however, this decrease was not significant.

FIG. 15 shows p75^ntr immunoreactivity in a rat forebrain showing heavy staining in the stria terminalis , basal ganglia/amgy and optic tract. Cortical staining was restricted to the infagranular portion. Vitamin D depletion appeared to drastically down-regulate p75^ntr immunoreactivity.

Sections were coded, randomised and assessed visually (P<0.001 Fisher's exact test).

FIG. 16(a) shows vitamin D depletion decreased the amount of NGF protein in the neonatal brain, ng/g protein (n=14 ± SEM) ^* (P < 0.015 unpaired t-test). . FIG. 16(b) shows vitamin D depletion decreased the amount of

GDNF protein.

FIG. 17 shows NT3 protein levels were unaffected by dietary intervention.

FIG. 18 shows BDNF and NT-4 protein levels were unaffected by dietary intervention.

FIG. 19 shows mRNA expression levels of neurotopin and receptors. p75ntr was significantly reduced in the depleted animals. Unless otherwise specified in all cases bar graphs indicate means ± SEM (n=10).

FIG.20(a) is an image of a Western blot showing no decrease in the Vitamin D receptor protein (VDR) in deplete (lanes 5-8), compared with control animals (lanes 1-4).

FIG. 20(b) shows semi-quantitation of VDR by densitometry. Bar graphs indicate means ± SEM (n=10).

FIG.21a shows total distance traveled in relation to locomotor and exploratory behaviour in the hole board apparatus. Female (open bars) and male (closed bars) rats in control, Day 0, Day 21 and Deplete all groups. Data are mean ± SEM. * P < 0.05, Day 21 vs. control.

FIG.21b shows a number of head dips in relation to locomotor and exploratory behaviour in the holeboard apparatus. Female (open bars) and male (closed bars) rats in control, Day 0, Day 21 and Deplete all groups. Data are mean ± SEM. * P < 0.05, Day 21 vs. control.

FIG.22a shows mean ± SEM percent time spent on open arms on the elevated plus maze. Data are presented for female (open bars) and male (closed bars) rats in control, Day 0, Day 21 and Deplete all groups. * P < 0.05, Day 21 vs. control.

FIG. 22b shows mean ± SEM number of arm changes on the elevated plus maze. Data are presented for female (open bars) and male (closed bars) rats in control, Day 0, Day 21 and Deplete all groups. * P < 0.05, Day 21 vs. control.

FIG. 23 shows mean ± SEM time spent investigating a conspecific in the social interaction test. Data are presented for female (open bars) and male (closed bars) rats in control, Day 0, Day 21 and Deplete all groups. * P < 0.05, Deplete all vs. control.

FIG. 24 shows acoustic startle responses to different auditory stimuli (70-120 dB) for control (A,B), Day 0 (C,D), Day 21 (E,F) and Deplete all rats (G,H). Data are shown for females (A,C,E,G) and males (B,D,F,H).

Data are presented for the mean ± SEM average amplitude of the startle response during a 200 msec window after stimulus onset.

FIG. 25 shows prepulse inhibition of the acoustic startle response for a second set of experiments. Data are shown for females (A,C,E) and males (B,D,F) in control, Day 0, Day 21 and Deplete all groups. The bars in each group represent the average response of 5 trials from nine different prepulse stimuli, varying by prepulse intensity (74, 78 or 86 dB; (left to right within each group; see data for Cont F group) and prepulse to pulse interval [8 msec (A,B), 32 msec (C,D), 256 msec (E,F)). The pulse was a 120 dB white noise burst presented for 40 ms above a 70dB background. The percentage PPI was calculated as [100 - (100 x startle amplitude on prepulse trial)/(startle amplitude on pulse alone trial)]. Note that a positive PPI score indicates inhibition of the startle response and a negative PPI score indicates facilitation of the startle response. FIG. 26A shows total time observed immobile in a forced swim test. Female (open bars) and male (closed bars) rats in control, Day 0, Day 21 and Deplete all groups. Data are mean ± SEM.

FIG. 26B shows total time engaged in escape behaviours in a forced swim test. Female (open bars) and male (closed bars) rats in control, Day 0, Day 21 and Deplete all groups. Data are mean ± SEM.

Table 1 : Doubly multivariate repeated measures analysis of variance on means of prepulse inhibition data.

Table 2: Mean body weights (+ SEM) for each Group at different Ages. Table 3: comparison of morphological features of control animals and animals born from a mother fed a vitamin D deficient diet.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method for producing a non-human animal having a modified behaviour and/or morphology, an animal having a modified behaviour and/or morphology and use of the animal for screening an active molecule or drug potentially useful for treating a disorder. Preferable, the animal is an animal model for a neuro-psychiatric disorder, in particular schizophrenia as exemplified herein.

The inventors have found that feeding a mother or dam a vitamin D deficient diet results in a pup having a change in behaviour and morphology when compared with a normal untreated animal. The behaviour and morphology change is consistent with a neuro-psychiatric disorder, including schizophrenia.

Studies herein also show that the change in behaviour and morphology in the pup may be influenced by feeding the mother a vitamin D deficient diet after giving birth (i.e. when the mother is nursing the pup) and feeding the pup a control diet (i.e. normal vitamin D levels in the diet) after weaning. Changing the vitamin D content ofthe diet ofthe mother after birth and pup provides a means for further modifying the behaviour and/or morphology of the animal. Accordingly, selecting the vitamin D content of the diet of the mother after birth and pup may be used to make an animal model for a particular disorder. It will be appreciated that a vitamin D deficient diet is a convenient means for reducing an amount of vitamin D in an animal, e.g. the mother and pup. However, other means for reducing vitamin D in the mother or pup may also be used. Accordingly, a vitamin D deficient diet is a preferred means for reducing an amount of vitamin D in the mother and pup. Definitions

Unless defined otherwise, all technical and scientific terms used herein have a meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any method and material similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, preferred methods and materials are described. For the purpose of the present invention, the following terms are defined below. As used herein, "neuro-psychiatric disorder" also refers to a neuro-developmental disorder including, for example, a disorder associated with increased dopaminergictone, schizophrenia, multiple sclerosis, anxiety, autism, Alzheimer's disease, Parkinson's disease, depression, mania, attention deficit/hyperactivity disorder dementia. As used herein, "vitamin D" may also refer to a vitamin D precursor, a related compound and vitamin D-like compounds. For example, 1,25 dihydroxyvitamin D₃, calcitriol, cholecalciferol, pre-vitamin D, and related synthetic analogues fall within the term "vitamin D".

A "deficient amount of vitamin D" refers to an amount of vitamin D that is less than a normal amount for an animal. A "reduced amount of vitamin D" may refer to a plasma concentration of 25 Hydroxy vitamin D in an animal that is less than normal. For example, in a mother rat, a reduced amount of vitamin D refers to a plasma concentration of 25 Hydroxy vitamin D that is less than about 15 ng/ml and a plasma concentration of 25 Hydroxy vitamin D in a neonatal rat that is less than about 30 ng/ml. In humans, a 25 hydroxyvitamin D levels equal or less than 50 nmol/L is consider clinical insufficiency and levels equal to or less than 38 nmol/L is consider clinical deficiency. A normal amount of vitamin D refers to a plasma concentration of 25 Hydroxy vitamin D in a mother rat that is at least about 15 ng/ml and a plasma concentration of 25 Hydroxy vitamin D in a neonatal rat that is at least about 30 ng/ml.

A "diet deficient in an amount of vitamin D, vitamin D precursor or vitamin D-like compound' refers to any diet comprising less vitamin D than is typically feed to a particular animal, including a diet without any detectable vitamin D. This includes a diet comprising less vitamin D than that required for normal health ofthe animal. A deficient vitamin D diet may reduce serum 25 Hydroxy vitamin D levels to less than about 30 ng/ml in a neonatal rat and less than about 15 ng/ml in an adult rat, or even less than about 5 ng/ml in a neonate and/or adult. A diet sufficient in vitamin D comprises an amount of vitamin D for normal health of the animal and preferably provides enough vitamin D so that plasma concentration of 25 Hydroxy vitamin D in an adult rat is at least about 15 ng/ml and a plasma concentration of 25 Hydroxy vitamin D in a neonatal rat is at least about 30 ng/ml. Likewise, a sufficient amount of vitamin D should prevent a diseased state such as rickets and abnormal bone development.

"Early development' includes any stage post-fertilization and pre-weaning; for example, embryonic and fetal development in utero.

"Normal Animal" refers to an animal that is representative of a normal population for the animal. A normal animal may include an untreated animal. "Normal behaviot" refers to behavior that is typical for a normal animal.

"Modified behaviot" includes abnormal behavior, which refers to behavior that is not typical for a normal animal. For example, behavior associated with a neuro-psychiatric disorder such as increased dopaminergic tone and symptoms of schizophrenia. Abnormal behavior includes hyperlocomotion, decreased social interaction and/or a change in pre-pulse inhibition.

"Modified morphology refers to a change in normal morphology and includes a change in brain morphology such as a change in ventricle size. Modified morphology also includes a change in expression of proteins, such as neurotrophins and receptors and a change in expression of nucleic acids encoding said proteins.

A "change towards normal behaviot³' refers to measurable change in behavior that is more similar or identical with normal behavior when compared with behavior before the change in behavior. For example, a change in behavior towards normal behavior may occur after administering a drug to the animal.

As used herein, "drug" also means "pharmaceutical composition" which comprises an active molecule or substance including a compound, typically in combination with a pharmaceutically-acceptable carrier. A molecule may be an active substance capable of causing a response, for example an active substance may be capable of modifying a behaviour of an animal. The modified behaviour may be a change from an abnormal behaviour, e.g a schizophrenic behaviour, towards a normal behaviour.

By "pharmaceutically-acceptable carrier, diluent or excipient" \s meant a solid or liquid filler, diluent or encapsulating substance. Drugs which may be used with the animal model include, for example, currently available anti-psychotic drugs such as haloperidol, clozapine, or others. These and other drugs are described in MIMS (MediMedia Australia Pty. Ltd, a Vivendi Universal Publishing Company; E-MIMS Version 4.00.0489, Vivendi University Health) and Goodman and Gilman's The Pharmacological Basis of Therapeutics (New York: McGraw-Hill, c2001, 10^th Ed. Editors, Joel G. Hardman, Lee E. Limbird, consulting ed. Alfred Goodman Gilman), both references are incorporated herein by reference.

The following notations are used herein for a first set of experiments shown in figures 1-20. A more detailed description is provided in the Example section. "Con" means controls (mothers, fathers and pups fed a control diet comprising 1 ,25(OH) vitaminD₃; "D0"means deplete fetus (mother on depleted diet (ie. without vitamin D₃); pups put on control diet at birth); "D30" means deplete weaning (mother on depleted diet; pups put on control diet when weaned at 30 days); "NoD" means deplete all (mother on depleted diet; pup on depleted diet until the end of the experiment at 10 weeks); "Norm" means normal control group was fed and housed similarly to the controls except that litter size was not culled to two animals; and "Late" means a control group which was of normal litter size and fed and housed similarly to the "Norm" group until 5 weeks of age when they were depleted of vitamin D similarly to the "NoD" group until 10 weeks of age.

Results shown herein are from experiments in relation to a method for making an animal model. The animal model is for a neuro- psychiatric disorder, in particular for schizophrenia. The study in particular examines effect of vitamin D depletion on the neuroanatomy of adult rats and on their behaviour.

The inventors have shown that a vitamin D-deficient rat is a useful animal model for a neuro-psychiatric disorder, including schizophrenia. The animal model exhibits a phenotype characteristic of the neuro-psychiatric disorder, schizophrenia. The phenotype includes: 1) hyperlocomotion, 2) decreased social investigation, 3) a change in pre-pulse inhibition when compared with a normal and 4) vitamin D depleted rats have larger ventricles than controls. The above behavior and morphological changes are examples of some measurable changes identified in the animal model and is not intended to be exhaustive of possible changes. Accordingly, a person skilled in the art would be able to select and test for other changes in the animal model that may correlate with a neuro- psychiatric disorder. Also, a reversal of a change associated with the animal model when administering a known drug to the animal model may further validate the animal model for a particular neuro-psychiatric disorder. Developmentally dependent change in pre-pulse inhibition in vitamin D depleted adults

Acoustic startle is a fast muscular twitch in response to sudden loud startling sounds [Koch, 1999], incorporated herein by reference. Magnitude of this response is attenuated if the startling sound is immediately preceded by a distinct non-startling sound. This attenuation is called prepulse inhibition (PPI) [Koch, 1999]. PPI is observed in animals including humans. Mechanisms of the brain underlying the mediation of PPI are not fully understood, but PPI is diminished in schizophrenic patients [Swerdlow, 1994], incorporated herein by reference, and anti-psychotic/anti- dopaminergic drugs restore PPI in patients as well as in dopamine-activated rats [Swerdlow, 1994].

In a first set of experiments, PPI was reduced by vitamin D depletion in an age-dependent manner. Although PPI may be useful as one criteria when characterizing a neuro-psychiatric disorder, this criteria is not essential to characterizing the animal model. Measuring PPI is only one possible test that may be used when assessing a behavior change. Accordingly, it will be appreciated that data in relation to a change in PPI may vary as shown herein without detracting from an importance of the animal model for a neuro-psychiatric disorder. The data indicates that there is a change in PPI when comparing the animal model and a control animal, however this change may differ between experimental conditions.

Animal models for schizophrenia may show a reduction in prepulse inhibition of startle response [Swerdlow, 1994; Koch, 1999]. The inventors show in a first set of experiments that vitamin D depletion during development and into adulthood reduces pre-pulse inhibition in rat. Furthermore, this reduction in pre-pulse inhibition is developmentally regulated: it is evident in animals aged 10 weeks old, but not 5 weeks. This effect at 10 weeks is not due to vitamin D deficiency acting solely on the adult brain since it is not present in animals depleted of vitamin D during the second 5 weeks of life. The effect at 10 weeks is also not solely due to vitamin D deficiency acting early in life since it is not present in animals depleted of vitamin D up until weaning at 4 weeks of age (D30).

These results indicate that vitamin D deficiency must be present both during development and in adulthood for the impairment of prepulse inhibition to be evident.

One difference between rat and schizophrenic human is a recognised genetic contribution to the disease. In the human low vitamin D would be acting on a mutated genetic background not present in the rat. Furthermore, the brain of a newborn rat is much less mature compared to a brain of a newborn human, accordingly, a critical window during which low vitamin D impact brain development in the rat would require additional postnatal depletion. From this, not being bound by theory, the inventors propose that a genetic predisposition to schizophrenia will be found in the vitamin D signaling pathway - either within the vitamin D pathway itself or those genes that it regulates.

Vitamin D3 Deficiency Impairs Pre-Pulse Inhibition (PPI) Startle testing revealed an age-dependent reduction in PPI evident only in the Deplete All group, NoD (FIG. 1). At 5 weeks of age (FIG.

1A and 1B) all groups of animals showed similar prepulse inhibition.

There was increasingly more inhibition with louder prepulse stimuli from 5 to 15 dB (decibels) above background sound levels. Statistical analysis revealed significant Group, Sex, Trial and Age differences (Table 1), eg. differences due to Vitamin D status (Group), between males and females

(Sex), due to a volume of prepulse (Trial) and when tested at Week 5 and

Week 10 (Age).

A Group x Age interaction was significant (Table 1) indicating that differences due to Vitamin D status were dependent on the week of testing. Visual analysis of the means indicates that main differences in the data occurred in the NoD group at 10 Weeks. Post-hoc analysis indicated that the NoD group was significantly different from all other groups at Week

10 (Table 1). Vitamin D3 Deficiency did not Affect Startle Response Sensitivity

It is possible that differences in prepulse inhibition may arise from differences in auditory sensitivity. Threshold of the startle response is related both to auditory sensitivity and muscular ability to respond. It was shown that animals in all groups were able to respond. In particular the Deplete All group (NoD), which had impaired prepulse inhibition, had larger responses than the other Groups when the responses were normalised for body weight (FIG.2). Accordingly, it appears that the amplitude ofthe startle response to low level sounds is a function of auditory sensitivity.

A comparison of auditory sensitivity was made by measuring the startle response magnitude at varying sound pressure levels from 65dB (background noise level) to 120dB (PPI test pulse level). There was no obvious difference among the groups at 5 Weeks (FIG. 3A) or 10 Weeks (FIG. 3B). Visual inspection of these curves indicates that all Groups had similar response thresholds of 90dB (FIG. 3). A MANOVA with Age, Group and Sound Pressure (dB) indicated no significant differences among the groups. Startle response amplitude There were no differences in body weight at 5 Weeks, but at 10 weeks the vitamin D depleted animals (NoD) were significantly smaller than the Controls (Table 2). When the startle response was corrected for body weight they were revealed to have larger startle responses relative to body weight (FIG. 4). There was no relation between amplitude of the startle response and degree of prepulse inhibition. There was no correlation between the mean amplitude of the startle response during a 120 dB test pulse trials with the mean levels of prepulse inhibition when all data were compared at 10 weeks. For prepulse stimulus amplitudes of 5dB, 10dB and 15dB the Spearman correlations (r) were -0.01 , -0.03, and -0.08.

These observations confirm that the impairment of prepulse inhibition was not due to an inability to respond to the acoustic stimuli. In other words, the animals could both hear and respond to the stimuli.

Such a symptom could arise from changes in excitability of neuromuscular circuitry, but at least is not explained in terms of simple variable such as serum calcium levels, which did not differ among the groups when measured at least in neonates (see FIG. 8b). It has been argued that reductions in prepulse inhibition can result from increased reflex excitability [Schicatano, 2000], but this was not evident in the present study. There was no correlation between amplitude of the startle response to the 120 dB SPL stimuli and magnitude of prepulse inhibition. This was true when data from all animals were compared, when data from each group were compared, and for individual animals (data not shown).

Vitamin D3 deficiency increases startle response latency

Latency of the startle response was significantly longer in the Deplete All group (FIG. 5). Among all groups there was a tendency for the amplitude ofthe startle response to increase with latency. A linear regression comparing mean amplitudes and latencies for the 120dB trials for all animals in all groups at Week 10 was significant (R = 0.60, Fι,ι₂5 = 69.216, p<0.001 ). This relation accounted for 35% of the variance in the data. A linear regression limited to the Deplete All Group gave a similar result (R = 0.65, Fι,ιβ = 12.112, p = 0.003), accounting for 38% of the variance in the data. There was contrary evidence that reflex excitability was reduced in the vitamin D depleted adults because these animals had significantly longer latencies to respond to the acoustic stimuli and there was a small, but significant trend for startle response amplitude to be positively correlated with startle latency. This is contrary to a usually observed reduction in latency associated within prepulse inhibition [Koch, 1999].

An increase in latency is of biological significance in terms of the animal model because a reduced latency is associated with reduced PPI. A reduced latency is also associated with fear and anxiety, which are associated with reduced PPI. An increased or higher latency is therefore significant because it is additional evidence that the reduced PPI observed in the NoD animals is not due to anxiety (which is further supported by a lack of difference in the open-field). An increase latency also confirms that the reduced PPI observed is not due to a simple increase in reflex excitability [Schicatano, 2000 ] since reflex excitability would be associated with a reduced latency. Open Field Behavior (anxiety test)

One explanation for the enhanced startle response in the vitamin D depleted rats could be an increase in anxiety, which is known to increase startle reflexes in animals and humans [Koch, 1999]. As an independent test for anxiety animals were tested in an open-field. Locomotion in the open-field test is reduced in anxiety in rats [Crawley, 1999]. Locomotion in an open field is influenced by anxiety induced by the novel, open environment.

In order to test a time-structure of movement during a 200 second test session, a distance moved was calculated for each 10 second time period (FIG. 7). A distance moved in each 10 second period was very variable throughout the test session with all animals showing variably alternating periods of faster and slower activity with all animals more active initially. The time-structure ofthe activity was similar in all groups. Statistical analysis shows that the NoD group were less active at 5 weeks, but no different at 10 weeks when compared to other groups.

When placed in the open-field females were more active than males and younger animals were more active than older animals (FIG. 6). There does not appear to be a large difference in locomotion in the open-field, neither in the total distance travelled nor in the distance travelled during different time periods during the test period. Nor was there any obvious difference in the trajectories ofthe animals during the open-field test - all animals spent the vast majority of their time close to the walls of the test chamber (data not shown). These observation suggest that the groups did not differ in their levels of anxiety. In summary the reduced prepulse inhibition observed in the vitamin D depleted animals was associated with an increased amplitude of startle response and an increased latency to startle, but no differences in open-field locomotion or startle habituation. Vitamin D3 deficiency did not affect habituation ofthe startle response

Habituation is a reduction in response upon repetitive stimulation. The test trials were organised so that the first 44 trials were the same as the second 44 trials. That is, for each sound pressure level, 4 trials were delivered in each half ofthe test session. For each animal, means were calculated for the responses in first 4 trials and in the second 4 trials for 100dB, 110dB and 120dB. As a measure of habituation, the mean of the second 4 trials was expressed as a percentage of the mean of the first 4 trials, for each sound pressure level. A repeated measures ANOVA indicated that there were no significant differences among the Groups indicating that Vitamin D status did not affect habituation. There was no effect of Age or Sex on habituation. Specificity of Behavioural Impairment

Several behaviours were measured during startle testing to investigate specificity of an impairment in prepulse inhibition. First, there were no differences in the amplitudes of auditory stimuli, which first evoked a startle response. This indicates that the animals did not differ in their ability to hear the stimuli. Second, there were no differences in habituation to the stimuli and testing procedure as measured by the change in startle response recorded for the first half of the session compared to the second half. Separate measurements of the open field locomotion indicates that the animals did not differ either in the total distance travelled in the test session or in the speeds travelled at different periods during the test session. Vitamin D Depletion and Physiological and Morphological Changes

Changes in Brain Morphology

Vitamin D-depleted pups have heavier bodies and brains when compared with controls, however, a ratio brai body remains similar to control animals.

Comparisons of brain and body weights were made on pups from a median litter size (n = 11). In a breeding programme a total of nine litters of this size were produced from both vitamin D depleted and control groups. Two males and two females were used in this study from each litter making a total of 36 pups from each group. Vitamin D-depleted pups were heavier at birth than the control animals (P < 0.0005). When males and females were compared separately this difference remained significant (6.42 ± 0.17 vs 5.85 ± 0.10g , P < 0.01 Males) and (6.06 + 0.16 vs 5.32 ± 0.15g , P < 0.005 Females). Brain weights were also heavier in the deplete group (P<

0.0001), and again this difference was retained when the same sex was compared (0.265 ± 0.007 vs 0.235 ± 0.004g, P < 0.001 males) and (0.254 ±

0.007 vs 0.221 + 0.004g, P < 0.001 females). The ratio of brain to body weight was not significantly altered between deplete and control groups. Therefore brain weight appeared to be directly proportional to body size indicating vitamin D depletion did not induce a distortion between the head and the rest of the body (Table 3). Interestingly compared with controls, brains of depleted animals were longer (P<0.002). This phenomena occurred in both males (6.55 ± 0.47 vs 4.88 ± 0.36 P<0.03) and females (6.05 + 0.24

vs 4.89 ± 0.34, P < 0.02).

Brains were bigger at birth in the vitamin D deplete group both in weight and length. Data on the effect of vitamin D on gross brain measurements is limited. Vitamin supplementation and depletion have predictable effects on body weight in rats during post-natal development; however at birth, body weight appears to be unaltered [Marya et al.,1989; Hallora and De luca 1979]. The fact that brain weight increased only in proportion to body weight did not necessarily imply that a gross abnormality was present in these animals. It was only when these brains were examined internally that a dramatic increase in lateral ventricle volume was revealed. The lateral ventricles of the deplete group were double that of the controls (Fig. 10). The magnitude of this increase remained even when corrected for the increased hemispheric volume seen in the deplete animals (FIGS. 11a and 11 b). Ventricular increases were restricted to the lateral structures.

Larger brains in the deplete animals did not necessarily lead to larger structures within the brain. In fact a possibility that the dramatic increase in ventricular volume may have induced a decrease in some major brain structures was considered. One obvious candidate is the thickness of the adjacent cortical mantle. Additionally the inventors measured the thickness of the largest white matter fiber tract in the brain, the corpus callosum as well as the hippocampus at the same coronal plane. In an absolute sense all of these structures were shown to be similar in treated and control groups. However when normalized for the slightly larger brain cross-sectional area seen in the deplete animals then the width of the neocortex was shown to be proportionally smaller (Table 3). Vitamin D deplete animals have larger lateral ventricles and proportionallv thinner cortices

In all measurements of lateral ventricle volumes, 3^rd ventricle and hippocampal areas, cortical width and corpus callosum thickness there was no significant difference between males and females in respective deplete and control groups, therefore male and female data was combined . As there was no significant difference in lateral ventricle volumes from either hemisphere, cortical mantle thickness and hippocampal areas are reported as the average for both hemispheres.

The most obviously altered structures in the vitamin D deplete animals were lateral ventricle volumes which were much larger in the deplete animals (P<0.01) FIGS. 10 and 11. This finding may have simply reflected the larger cortical hemispheric volume in the depleted animals (P< 0.001). All brain structures were therefore reanalysed correcting for either hemispheric volume or cross-sectional area. When corrected for the hemispheric volume, lateral ventricular volumes remained on average twice as big in the deplete animals (P<0.05) FIG. 11 (b). An increase in the volume of such a large internal structure such as the lateral ventricles may indicate tissue loss elsewhere. We therefore examined several major structures for apparent tissue loss. Ofthe remaining structures examined the embryonic cortical mantle and corpus callosum were of a similar thickness in both controls and deplete animals, but when examined as a proportion of the larger cross-sectional areas present in the deplete animals the cortex but not the corpus callosum was shown to be significantly thinner (P< 0.01). These results are collated in Table 3.

Collectively these morphological alterations are consistent with a developmental brain disturbance. Ventriculomegaly is the most commonly reported pathological abnormality in schizophrenia [Lawrie and Abukmeil 1998] a disease in which the wealth of evidence for a developmental contribution now seems compelling [Weinberger 1986]. Enlarged ventricles are also present in other diseases believed to have a developmental component, i.e. autism [Gaffney et al., 1989].

A reduction in cortical mantle width has also been proposed as a prominent pathological marker for schizophrenia [Selemon 1995]. Reports of decreased cortical width in patients have been associated with decreased neuropil and increased cell density [Selemon 1995 and 1998]. In the cortical region assessed in this study cell density was unaltered in the deplete group. This however is not inconsistent with an alternative mechanism for altered cortical neuropil density in schizophrenia i.e. synaptic pruning which is a late post-natal maturation event [Pettegrew et al. 1997].

These findings indicate that neonatal Vitamin D depletion can lead to changes in brain morphology also seen in schizophrenia (ventricular enlargement) and indicate a developmental mechanism via which this can occur (changes in cell proliferation and nerve growth factor signaling). Ventricular enlargement represents the most robust pathological finding in the brains of schizophrenics. The fact that ventriculomegaly can be induced experimentally in a developing animal strengthens the inference that this feature in patients is a direct result of altered brain development in utero rather than a subsequent degenerative event. Vitamin D depletion did not alter cell density in any brain region examined (FIG. 12). Mitosis and apoptosis Vitamin D depletion increased the number of mitotic cells

Vitamin D depletion did not alter cell density in any brain region examined (FIG. 13). However, vitamin D-depletion appeared to selectively increase a percentage of cells in the brain that were mitotic. Between 0.5-0.1 % of the cells in the neonate were still within the cell cycle depending on the region analysed. Mitotic rates were significant in all regions except the cingulate (FIG. 13).

The percentage of cells with nuclear morphology indicative of apoptosis was much lower, between 0.001 and 0.035%. The basal ganglia/amygdala was readily distinguished from the other three regions shown in that the number of apoptotic cells was clearly much greater perhaps reflecting delayed ongoing development in this region (see FIG. 14). Although the ratio of apoptotic to non-apoptotic cells was reduced in all regions within the depleted group this was not significant (FIG. 14). For all measurements - cell density, apoptosis and mitosis ratios - hemisphere asymmetry and gender were not significant variables.

These morphological changes were not the result of some general glial or neuronal alteration. Neither GFAP or neurofilament content appeared to be affected as a result of dietary modification (data not shown). Cell density was unchanged in every brain region investigated. Additionally the thickness of the largest white matter structure in the brain was unaltered indicating no general glial cell loss.

In addition to these morphological alterations this study provides some of the first evidence in vivo that in keeping with the role of vitamin D in other organs this vitamin may be a potent regulator of cellular maturational processes in the developing brain. Four specific brain regions were selected for cellular analysis. Firstly the hypothalamus was chosen due to its central role in endocrine function in the brain. The cingulate gyrus was selected as a representative circumscribed cortical region in addition to the often-reported pathologies in this nucleus in schizophrenia (Benes 1998). The dentate gyrus was chosen as it is a portion of the brain that has long been recognized as a site for neurogenisis (Altman and Das 1965) thereby maximising the possibility of assessing variability in cellular differentiation and proliferation. Finally the basal ganglia/amyg was investigated as dopaminergic abnormalities have often been linked with schizophrenia and other developmental conditions.

The data reveals that when vitamin D is absent there is an alteration in cellular processing consistent with a failure of cells to opt out of the cell cycle and begin to mature and differentiate as well as a tendency towards a reduction in normal programmed cell death. Essentially the pattern was towards higher mitotic rates in all brain regions investigated. This was significant in all areas except the cingulate gyrus. Consistent with the picture of an unimpeded cell-cycle was the non-significant trend towards lower rates of apoptosis in all four brain regions but this did not reach significance. Why mitosis was not significantly elevated in the cingulate is unknown. The fact that it also contained the lowest percentage of apoptotic cells of any region investigated may reflect a greater maturity of this nucleus at birth.

Such a picture is entirely consistent with the known actions of vitamin D in numerous tissues. 1 ,25-dihydroxyvitamin D₃ has been shown to down regulate cyclins which are proteins that govern transition points through the cell cycle (Laud et al., 1997). This may therefore be one mechanism for vitamin D's antiproliferative effects. Apart from the well-documented effects on bone, vitamin D exerts an antiproliferative action on cells throughout the body i.e. heart (O'connell et al., 1997); gut (Menard et al., 1995); kidney (Weinreich et al., 1996). Additionally there is abundant evidence that vitamin D promotes cellular apoptosis in a variety of malignant cells such as gliomas (Naveilhan et al.,1994; Baudet et al., 1998); breast cancer cells (Mathiasen et al., 1999); and colon cancer cells (Vandewalle et al., 1995). In an analogous study in adult rats it is interesting to note that vitamin E deficiency enhanced neurogenisis in the dentate gyrus of adult rats (Ciaroni et al., 1999). This suggests that there may be a general role for fat-soluble vitamins as inhibitors of neuronal proliferation.

Taken together these findings lead to a hypothesis that in the absence of vitamin D its antiproliferative and proapoptotic actions are diminished leaving a significantly greater number of cells in the cell cycle. The presumed outcome from increased cell production and reduced cell loss in the absence of an increase in cell density would be a larger brain. If the general pattern observed in the four brain regions examined is extended throughout the brain then this may provide an explanation for the increase in brain weight, size and length observed in the vitamin D deplete animals. Immunohistochemistry

Vitamin D depletion failed to affect the distribution of all high- affinity neurotrophin receptors in the neonatal brain. Distribution ofthe low- affinity non-selective neurotrophin receptor p75^πtr in ratforebrain; however, it was profoundly influenced by vitamin D status. p75^ntr immunoreactivity was most striking in the stria terminalis (FIG. 15). Staining was also present in the endopunduncular nucleus within the basal ganglia/amygdala and the infagranular portion of cortical plate and the optic tract. In all regions p75^ntr staining was drastically reduced in the deplete animals. This was established by coding and randomising sections followed by visual assessment (PO.001) (Fig. 15).

The low affinity, non-selective, neurotrophin receptor p75^ntr was examined immunohistochemically at single medial sections throughout the neonatal brain. Surprisingly, vitamin D depletion virtually abolished p75^ntr immunoreactivity (FIG. 15). p75^ntr was found in the stria terminalis, basal ganglia/amy, optic tract and infagranular portion of cortical plate. The localization of p75^ntr to these regions is of particular relevance given the distribution ofthe VDR in the brain. The stria terminalis has consistently been shown to contain the greatest density of uptake sites for 1,25- dihydroxyvitamin D₃ in rat brain (Stumpf et al., 1982; Stumpf and O'Brien 1987). The basal ganglia/amy also contains VDRs (Veenstra et al., 1998; Prufer et al., 1999), which are dynamically expressed in development. The degree of co-expression of these receptors at the cellular level in these regions is unknown and will be explored in the future. Vitamin D has been shown to directly regulate p75^ntr in glioma cells (Naveilhan et al., 1996; Baas et al., 2000). The greatly diminished immunohistochemical response for p75^ntr therefore in the absence of the vitamin is entirely consistent. Immunohistochemistry studies revealed that ofthe high affinity neurotrophin receptors, trk B, the receptor selective for BDNF and NT-4 was the most widely expressed throughout the brain. No qualitative change in either density or location of trk A, B or C was visually apparent in these studies as a result of dietary modification (data not shown). FIG. 19 shown that there was no significant change in trkA, B or C message levels as determined by rtPCR.

In the neonatal cortex trkA and p75^ntr immunoreactivity appeared to be present in distinct bands, trk A was restricted to the outer supragranular layer and p75^ntr appeared restricted to the inner infagranular portion. At later developmental stages p75^ntr has been shown to be a proapoptotic signal to cells when coexisting trk expression is absent (Rabizadeh et al., 1993; Barrett and Bartlett 1994; Friedman 2000). Therefore at this late embryological stage, cells within the infagranular portion ofthe developing cortex may be tagged for programmed elimination. The dramatic reduction in p75^ntr immunoreactivity in deplete animals may therefore impede this elimination, consistent with the trend towards a diminished rate of apoptosis throughout the brain. Quantitative protein analysis

Vitamin D depletion decreased free NGF protein levels by 17% (P < 0.05) (FIG. 16a) and decreased GDNF protein levels by 25% (FIG 16b). The protein levels ofthe other three neurotrophins investigated (BDNF, NT-3 and NT-4) were unaltered (FIGS. 17 and 18). The ratio of neurofilament protein or GFAP relative to total brain protein was unaltered implying no selective alteration in neuronal or glial density (data not shown).

A decrease in NGF protein in the deplete neonatal brain is consistent with the well-described role vitamin D has in promoting NGF levels in vitro (Neveu et al., 1994; Wion et al, 1991; Musiol and Feldman 1997). The inventors, however, are unaware of any model either in vitro or in vivo that has described this corresponding inverse relationship. NGF plays a crucial role in neuronal differentiation in post-mitotic neurons (Thoenen 1991). The data has shown a decrease in the expression of both its protein and a trend towards a decrease in its message. The resultant outcome in the neonatal brain can only be speculated upon but presumably any NGF dependent maturational processes will be impaired. Given that role played by this neurotrophin in particular is likely to be more important in the post-natal animal it would be of interest to examine the brains of deplete animals at later time periods. It is interesting to note that a link between ventriculomegaly in human fetuses and lower levels of NGF in amniotic fluid has been made (Marxet al., 1999) consistent with the morphological findings reported here. Essentially no data exists examining the effects of vitamin D on the expression of the three other major neurotrophin proteins BDNF, NT-3 and NT-4. These compounds were all shown to be unaltered by vitamin D depletion. They were however quantitatively almost an order of magnitude greater in the neonatal brain than NGF consistent with the knowledge that they are generally expressed at an earlier developmental stage than NGF (Fukumitsu et al., 1998; Ip et al., 2001).

Depletion of vitamin D did not appear to affect the apparent density of its receptor (FIG. 20). The VDR has been shown to upregulated by 1 ,25-dihydroxyvitamin D₃ in a number of tissues (Darwish and DeLuca 1993). This mechanism is believed to be via ligand-induced stabilization of the receptor rather than due to increased synthesis (Wiese et al.,1992). In the absence of the steroid therefore it could be hypothesised that the VDR might be subject to greater degradation. However, a western blot data shown in FIG. 20a would tend to discount this. Neurotrophin and neurotrophin receptor mRNA

FIG. 19 shows that vitamin D depletion did not affect the mRNA expression of all four major neurotrophins. The level of NGF mRNA was increased, but this was not significant. Ofthe three high affinity neurotrophin receptors trk B expression was elevated and trk A mRNA levels were decreased, but not significantly.. In parallel with the greatly diminished immunohistochemical expression of p75^NTR, there was a 30% decrease in expression of p75^NTR mRNA in whole brain extracts (ANOVA, P<0.01 , n=10). Decreases in the expression of the low affinity neurotrophin receptor, p75, observations are consistent with the known effect of vitamin D in promoting NGF expression in vitro [Neveu, 1994; Musiol and Feldman, 1997; Wion, 1991]. There was also a significant decrease in the expression of glial cell line-derived neurotrophic factor (GDNF) in the brain of vitamin D depleted neonatal rat pups.

Regarding the effect of vitamin D on neurotrophin transcript expression there is a wealth of data indicating the vitamin upregulates NGF transcripts (Wion 1991; Neveu et al., 1994; Musiol and Feldman 1997). In absence of vitamin D, there does not appear to be any significant change in NGF transcript levels (FIG. 19). One study has also reported the up- regulation of NT-3 and down-regulation of NT-4 mRNA in rat primary astrocyte culture in the presence of vitamin D (Neveu et. al., 1994). Data presented herein are consistent in that an inverse relationship, i.e. a slight down-regulation of NT-4 transcript, is shown in the deplete animals, however, these changes were not significant

In summary, an absence of vitamin D in utero produced a host of developmental changes in the neonatal brain. These were observed at the gross anatomical, cellular, protein, receptor and transcript levels. The findings are consistent with known effects of this vitamin in other organs and in neural tissue in vitro, namely that decreased vitamin D diminishes apoptosis, leads to a decrease in important trophic factors such as NGF and causes a down-regulation of p75^ntr immunoreactivity, all factors that vitamin D₃ is known to promote. Vitamin D is also known to inhibit some cellular processes. The data indicate that in a reciprocal fashion its absence allows these processes i.e. mitosis, to occur unimpeded. All these findings may be interrelated and such alterations may be crucial to the mechanisms underlying the ventricular and brain hypertrophy observed in the vitamin D depleted neonates.

Vitamin D depletion as a model for schizophrenia

A number of authors have emphasised several criteria for assessing validity of animal models of human brain disease [Belzung, 2001 ; Lipska, 2000]; these include "face validity", a degree to which responses in the model are identical to those in the human; "construct validity", a degree to which a model reconstructs the aetiology and pathophysiology of the disease; and "predictive validity", a degree with which statements about the human disease can be made from an understanding of the model. The animal model has "face validity" in that there is an alteration in prepulse inhibition, in particular there may be an impairment of prepulse inhibition and the onset of the behavioural impairment in early adulthood. A vitamin D-deficient animal model has a deeper "construct validity" in that it arises from human epidemiological data and reconstructs the impairment of prepulse inhibition seen in patients. It is also based on a "neurodevelopmental aetiology", as are current theories of schizophrenia based on neuroanatomical and other evidence from human patients.

The vitamin D-deficient animal model has "predictive ability" for future treatments, genetic factors and aetiology of schizophrenia. The animal model suggests that candidate genes for schizophrenia risk may be found among vitamin D related genes: within the vitamin D regulatory pathways or within pathways regulated by vitamin D.

According to Lipska and Weinberger [Lipska, 2000] innovative models will have the following goals: "(1) to test the plausibility of theories derived from emerging research data about the disorder; (2) to probe the explanatory power of new biological findings about the disorder; (3) to uncover mechanisms of schizophrenia-like phenomena; and (4) to suggest new potential treatments". According to these criteria, the animal model is innovative: (a) as described herein it consistent with low vitamin D in humans as a risk factor for schizophrenia; (b) it can be used to investigate new biological findings such as an involvement of genes revealed by recent DNA microarray studies [Hakak, 2001 ; Mimics, 2000]; (c) the model is revealing fundamental mechanisms of brain development which may be contributing to the observed differences in adult brain and behaviour; and (d) as indicated above the animal model may be useful in identifying directions for new treatment options. The potential to extend this type of research with the use of knock-out mice (looking for gene-environment interactions) and gene expression profiling (cross-referencing altered gene expression in animal experiments with gene expression studies in schizophrenia versus well controls) offers powerful new tools to the neuroscience community. Role of the animal model in schizophrenia A complete hypothesis for a cause of schizophrenia should be able to account for effects of neuroleptic drugs on symptoms of a disease as well as changes in neurotransmitter systems that underpin effects of a drug. The animal model may be used to study developmental changes wrought by low levels of vitamin D that may ultimately lead to subtle changes in cytoarchitecture and neuronal function that impact a wide range of neurotransmitter systems and neurotrophic factors with consequent effects on behaviour. A test for this is to determine whether a neuroleptic drug can alleviate observed altered behavior of the animal model, such as hyperlocomotion, decreased social interaction and a change in prepulse inhibition.

Vitamin D and Vitamin D Receptors

The active form of Vitamin D (1,25 dihydroxyvitamin D₃, calcitriol, cholecalciferol) is a steroid hormone deriving from an action of sunlight on 7-dehydrocholesterol in skin of an animal or person, followed by two separate hydroxylations, first in the kidney and second in the liver. Vitamin D binds its receptor (Vitamin D Receptor, VDR) to activate transcription. To initiate transcription VDR forms a heterodimerwith retinoid- x-receptor (RXR) and several other proteins before binding to the vitamin D responsive elements (VDRE) in the promoter region of many genes, including many of those expressed in the brain. It is interesting to note that the RXR family of transcription factors also heterodimerises with the Vitamin A receptor family (RAR or retinoid receptor). Vitamin A (retinoic acid) is known to induce the formation of several parts of the nervous system, including the forebrain, and has been suggested to play a role in the vulnerability to schizophrenia [LaMantia, 1999; Goodman, 1998]. Mice lacking both RAR and RXR have reduced levels of expression of the dopamine D2 receptor in the striatum [Samad, 1997] and have impaired locomotor behaviour [Kretzel, 1998] . Importantly the dopamine D2 receptor gene is regulated by RAR-RXR heterodimers and it is suggested that transcription of this gene may also be regulated by Vitamin D [Samad, 1997]. This hypothesis remains to be tested in a mouse lacking VDR but a behavioural phenotype for this mouse is not described [Yoshizawa, 1997]. Description of changes after Vitamin D depletion during embryogenesis has not been previously reported, although there is accumulating evidence that Vitamin D may be involved in brain development. Vitamin D may act on the developing brain via its effects on the neurotrophin signaling pathway which regulates neuronal survival and death (see below). Additionally there are other growth factor signaling pathways with which Vitamin D signaling interacts such as the transforming growth

factor-β pathway [Yanagisawa, 1999], and non-receptor protein kinase

pathway [Gniadecki, 1998]. Thus there is great scope for Vitamin D to act on brain development in a cell- and tissue-specific manner, depending not only on the expression of VDR-target genes, but also with interactions with other growth factor signaling pathways. Vitamin D receptors (VDR) are widely distributed throughout the embryonic brain prominently in the neuroepithelium and proliferating zones [Veenstra, 1998]. Expression is not confined to these regions; VDR is expressed widely in the adult brain in temporal, orbital and cingulate cortex, in the thalamus, in the accumbens nuclei, parts of the stria terminalis and amygdala and widely throughout the olfactory system. It was also expressed in pyramidal neurons of the hippocampal regions CA1, CA2, CA3, CA4. [Veenstra, 1998; Prϋfer, 1999].

Curiously, it bas been shown that dopamine and the dopamine agonist SKF38393 can activate the Vitamin D Receptor (VDR) in absence of 1 ,25 dihydroxyvitamin D (Matkovits and Christakos, 1995, Mol Endocrinol 9 232). Because the VDR and dopamine receptor are co-localized in areas of interest to schizophrenia research (central nucleus ofthe amygdala, reticular nucleus of the thalamus and ventromedial nucleus of the hypothalamus), agents that impact on dopaminergic neurotransmission (e.g. antipsychotic medication) may influence the activation of VDR-mediated regulation of neuronal genes. Not being bound by theory, disruption of brain development associated with vitamin D depletion may influence this ligand-independent activation of the VDR in the brain. Vitamin D and schizophrenia

Although the animal model herein described may be consistent with some known hypothesis of schizophrenia, none of the aspects of the invention are bound to any particular hypothesis. Also, the animal model may be characterized by only some behaviors associated with a neuro- psychiatric disorder and need not display or be consistent with all known behaviors for a particular neuro-psychiatric disorder for the animal model to be useful.

In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples. First set of experiments

EXAMPLE 1 Animals Sprague Dawley rats were used from the Central Animal

Breeding House, University of Queensland, Queensland, Australia. Animals were kept and raised on a 12:12 hr light cycle, lights on at 08:00. Animals were divided into 5 groups depending on their vitamin D and rearing status, with equal numbers of males and females in each group, where possible. At birth the litter sizes were culled to two, except in the Normal litter control group (below).

All experiments were approved by the Griffith University Animal Ethics Committee and conducted under the animal experimentation guidelines of the National Health and Medical Research Committee of Australia.

EXAMPLE 2 Vitamin D depletion Rats were fed a Vitamin D-deplete diet (Dyets Inc) and housed under incandescent light. On this regime, plasma levels in adults of 1 ,25(OH) vitaminD₃ are negligible after 3weeks (FIG. 8a and 9). Females and males were kept on this diet for 6 weeks and then mated. This feeding regime was maintained throughout pregnancy and after birth for pups and mothers where appropriate. Control rats were fed an identical diet except that it contained 1 ,25(OH) vitaminD3 (Dyets Inc) and housed under fluorescent lighting in a different room. On this regime plasma levels of 1,25(OH) vitaminD₃ were in the normal range (FIG. 8a and 9).

Rats were depleted and repleted at different periods during development: Controls (mothers, fathers and pups fed the control diet; Deplete Fetus (pups put on control diet at birth); Deplete Weaning (pups put on control diet when weaned at 30 days); Deplete All (deplete until the end of the experiment at 10 weeks); Late (depleted from weeks 5 to 10). An additional Normal control group was fed and housed similarly to the Controls except that litter size was not culled to two animals, to control for possible litter size effects on later behaviour.

Preliminary experiments indicated that vitamin D depletion resulted in an increased number of deaths during development, especially in male rats between 5 and 10 weeks of age. To guard against hypocalcaemia during this period these animals were supplemented with injections of calcium (2 ml Hartmann's Solution) twice weekly after weaning. Plasma calcium levels are shown in FIG. 8b. EXAMPLE 3

Behavioural analysis

At 5 weeks of age, and again at 10 weeks, all animals were given two behavioural assessments: (1) startle behaviour and (2) open field behaviour. All animals were tested between 10:00 and 16:00. Each animal was tested for open field behaviour followed by startle testing. Animals were not habituated to the procedures prior to the open field test but were habituated to the startle chambers for 5 min immediately prior to the commencement of testing.

EXAMPLE 4 Startle behaviour

Startle testing was performed using a four chamber system (SR-LAB; San Diego Instruments, San Diego, CA, USA). The system consisted of four startle chambers individually housed inside a sound- attenuating box with computer controlled delivery of sound stimuli and computer data logging of the startle response. Sound was delivered by a speaker mounted in side the sound-attenuating box. The system was housed in a dedicated room which allowed testing to proceed with minimal external ambient noise. The startle chambers were Plexiglas cylinders (8.7 cm internal diameter) mounted on a Plexiglas stand (12.5 cm²) within the sound- attenuating box. Animal movement was recorded as movement of the cylinder by a piezoelectric transducer attached to the top ofthe cylinder. The magnitude of movements were equalised between the startle chambers (SR- LAB Startle Calibration System) and sensitivities adjusted for testing 5 week (high sensitivity) and 10 week (low sensitivity) animals to ensure the responses were kept within the scale of the system. The use of individual chambers were balanced across all experimental groups and each chamber was washed with hot water and dried between each animal to reduce possible effects of odor signals released under stress [Mackay-Sim, 1980]. Sound levels were measured using a sound level meter and calibrated and equalised across all chambers using the software supplied (San Diego Instruments).

Startle testing was carried out with the animal inside the Plexiglas startle chamber in the dark with a background noise level of 65 dB controlled by the computer. Each test session was preceded by a 5 min habituation period. A test session comprised 88 stimuli and responses. A computer controled timing and delivery of sound pulses and data acquisition. Some trials consisted of single sound pulses only (startle trials) while others consisted of test sound pulses preceded by another sound pulse (Prepulse trials). Startle trial sound pulses (30 ms duration) were delivered at 65dB (background level), 70dB, 75dB, 80dB, 90dB, 100dB, 110dB and 120dB. Prepulse trials included a prepulse sound of 70dB, 75dB or 80dB preceding by 50mS a test sound of 120dB. In each session all trials were repeated 3 times, presented pseudo-randomly with a variable inter-trial interval such that the inhibition trials are distributed throughout the session. The inter-trial interval varied from 7 to 23 s, averaging 15 s, with different inter-trial intervals for each startle and Prepulse trial repeat.

The response to each startle and prepulse trial was recorded and for each animal the average response in each trial was calculated. Prepulse inhibition was calculated as the percent reduction in response in the Prepulse trials compared to the response to the 120dB sound delivered in the Startle trial.

Prepulse inhibition is thought to be a reflection of sensorimotor gating [Koch, 1999; Swerdlow, 1994; Swerdlow, 2000], although its magnitude can also be altered by changes in reflex excitability [Schicatano, 2000]. As a measure of indirect effects of vitamin D depletion on reflex excitability and muscle strength, startle responses were also compared after correcting for body weight. EXAMPLE 5

Open field behaviour

The effect of vitamin D depletion was also measured on locomotion in the open field as an assay of a more general behaviour related to anxiety [Koch, 1999] not related directly to sensorimotor gating. Open field behaviour was measured in a cylinder (90cm diameter, 60 cm walls) with black walls and base. It was lit by a 25W incandescent lamp suspended 70 cm above it. The animal was placed in the centre of the open field and its locomotion videotaped from above. The open field was wiped clean between animals using hot water and clean paper.

Locomotor behaviour was analysed digitally using the videotape as input into a frame-grabber card and using image analysis software (Optimas). The videotape was digitised at a rate of one frame per second. In each frame the position of the (white) animal was located in the (black) open field using its relative brightness and the thresholding function of the software. The distance moved between successive frames was calculated for 200 consecutive frames. From these data were calculated cumulative distance moved in 200 s and distance moved in each 10 s time period. EXAMPLE 6

Data analysis

Prepulse inhibition of startle and threshold of startle were analysed using a multivariate, repeated measures, mixed model analysis of variance with Group, Age, Sex and Trials as main effects. Data was expressed as mean and SEM. For statistical analyses data was normalised by log transformation where necessary.

EXAMPLE 7 Testing "Vitamin D" level in blood

As used herein, vitamin D depletion or reduction is meant in relation to vitamin D as generally referred to herein. In particular an active form of the steroid 1 ,25 diHydroxy vitamin D₃ and measured 25 Hydroxy vitamin D serum levels. 1 ,25 diHydroxy vitamin D₃ is not measured directly due to numerous assay and sensitivity problems. Instead an almost inactive (1 ,000 fold less biologically active) form 25 Hydroxy vitamin D is measured. This form is measured because it is far more technically easy to measure [Jongen, 1984]; is the most stable form of the vitamin and is the best indicator of overall vitamin D status in the animal reflecting D₃ from both dietary and UV_B radiation [Hollis, 1997].

A Diasorin radioimmunoassay kit was chosen as it had the best sensitivity of available methods and is the most widely used method to assess Vitamin D status in Australian pathology labs. The kit was used in accordance with the manufacture's instructions, incorporated herein by reference.

EXAMPLE 8 Animals used for Histology

Female Sprague-Dawley rats were fed a prepared diet free of sources of vitamin D (Dyets Inc CA USA), but with normal calcium and phosphorous. Animals were housed under a 12-hour light/dark cycle free of UVB light (excluding 290 - 315nm). After 6 weeks serum vitamin D depletion was confirmed prior to mating using a commercial RIA (Diasorin MN USA) for 25-OH D₃ which provides the best overall indicator of vitamin D status from both dietary and environmental exposures [Hollis, 1997]. The resulting dams were housed under these conditions until the birth of the pups. Control animals were kept under standard lighting conditions and were supplied with standard rat chow containing vitamin D (Dyets CA). To allow a uniform treatment of tissue, matings with vitamin D normal males were staggered in 6 waves of 10 dams (4 control and 6 Vitamin D depleted).

Because infant size may be inversely proportional to litter size only neonates from litters of 11 - 14 pups were selected for this study. Within 12 hours of birth 3 newborns of each sex were collected and birth weight recorded. The pups were killed by decapitation performed so that the brain stem was dissected in the same position for each animal. The brains were carefully removed, weighed and prepared for either histology, protein or RNA analysis according to the protocols described below.

Blood was also collected from neonates for 25-OH D₃, calcium and cholesterol assays. The 25-OH D₃ and Ca²⁺ status of the neonates and dams is shown in FIGS. 8 and 9. Note that neonatal Ca²⁺ levels are even higher in the deplete animals even though 25-OH D₃ levels are severely depleted (P<0.05). To address concerns that a virtual elimination of vitamin D from the diet may affect other cholesterol-based molecules, plasma cholesterol levels were measured in deplete animals. Plasma cholesterol levels found to be unaltered from controls (data not shown).

EXAMPLE 9 Histology and immunohistochemistrv

Once removed and weighed, brains were fixed for two hours in freshly prepared 4% paraformaldehyde/PBS, thoroughly rinsed, cryoprotected in 0.32M sucrose, embedded with cryostat mounting medium and frozen. Sections were cut on a IEC cryo-microtome (8 urn thick), thaw-mounted on pre-coated slides (Superfrost Plus, Lomb scientific) and stored at -80°C until use. Sections were stained in a series of 1 in 15 for nissl substance using cresyl violet. Sections were selected at the same medial coronal plane for immunohistochemical investigation. Individual antigens were visualised as follows. Endogenous peroxidase activity was quenched by incubating sections in 0.3% fresh H₂0₂ in either PBS (when the antigen was located on the outer surface ofthe membrane) or pure methanol (when the antigen was cytoplasmic, nuclear or located on the inner surface ofthe membrane) for 30 minutes. Non-specific staining was blocked by incubation for 1 hr with non- immune serum, appropriate for the secondary antibody, at a dilution of 1 : 10 in PBS containing 2% bovine serum albumin and 5% non fat dry milk. Sections were then incubated for 1 hr with primary antibodies diluted in the same blocking solution. Polyclonal anti-trk A, B and C antibodies (Santa Cruz Ca), polyclonal anti-vitamin D receptor (VDR, Santa Cruz), monoclonal anti- proliferating cell nuclear antigen (PCNA), (DAKO, CA) and monoclonal anti- p75 antibody (mc 192, Neubody, Flinders University, Australia) were used at the concentrations of 2 μg/ml, 2 μg/ml, 8 μg/ml and 10 μg/ml respectively. Negative controls were performed by omitting the primary antibody or by a 2 hour pre-incubation of the primary antibody with a five fold increased concentration ofthe corresponding blocking peptide. Sections were washed in PBS, incubated for 1 hr at room temperature with the appropriate biotinylated secondary antibody using Vectastain kits (1 :200, Vector labs, Burlingame, CA), washed in PBS, incubated for 1 hr in PBS containing 2% bovine serum albumin in the avidin-biotin-horseradish peroxidase complex (1 :50, Vector labs), rinsed and incubated for 5 min in Tris-HCI (0.05 M, pH 7.6) containing the chromogen diaminobenzidine (DAB, 0.05%) and H₂0₂ (0.004%). Slides were dehydrated and mounted in Depex. Photographs were taken on an Olympus microscope with a digital camera (Apogee Instruments) and Optimas software.

EXAMPLE 10 Brain Morphology

Brain area and length measurements were made from digitised section images (x200) that were analysed using NIH image software. Cortical and ventricle volume estimations were made from randomising a series (1 in 15) of sections through the whole structure under consideration. Volume estimations were then made using Cavalieri's principal (V = ∑APt), where V

is the volume ofthe structure; ∑ A is the sum ofthe cross-sectional areas; P is the inverse of the sample fraction; and t is the section width.

Other morphological observations were made on single sections using well-defined landmarks. Hippocampal and 3^rd ventricle area measurements were made on a series of three single sections at a level consistent with the anterior portion ofthe thalamus, the widening ofthe optic chiasm and immediately posterior to the decussation ofthe corpus callosum. Cortical width and callosum thickness measurements were made on a series of three single forebrain sections at the level of the decussation of the anterior commissure.

EXAMPLE 11

Apoptosis. mitosis and cell density counting

For these studies, sections from 10 control and 10 vitamin D deplete neonates with an equal partition between genders were used. For each animal, three brain sections were assessed. On each section, four specific regions from both hemispheres were examined: -the cingulate, the dentate gyrus, basal ganglia and the hypothalamus. To establish the presence of apoptotic nuclei sections were dried and incubated in the dark for 30 minutes in PBS solution containing 1 μM of bisbenzimide (Hoechst Blue 33258, Sigma). To establish whether a cell was undergoing mitosis, 3 sections from the immediately posterior slide were immunohistochemically stained for PCNA. Sections were viewed (800X) using an immersed lens and cells with strong immunochemical staining for PCNA (FIG. 13b) or obvious nuclear fragmentation counted. Stained cells in each individual field, as well as the total number of cells (phase contrast) were counted from digital images using NIH image software. Quantitation was performed blind to group status by two independent observers. Each count was expressed as a percentage relative to total cell number and cell density was expressed per unit of surface (e.g. % apoptotic cells/mm²). EXAMPLE 12

Protein analysis

Brains were homogenised using glass potter apparatus in 1 ml of cold 0.32M sucrose containing a protease inhibitor cocktail (Boehringer Mannheim) and 1 mM phenylmethylsulfonyl fluoride, and were stored frozen. The protein concentration of homogenates was estimated using a Bradford assay. Total brain vitamin D receptor density was qualitatively assessed by western blot using the same polyclonal antibody used for immunohistochemistry. Brain neurofilament and glial fibiliary acidic protein (GFAP) content was assessed by western blot using antibodies from Sigma and DAKO respectively. Free levels of nerve growth factor, (NGF); Brain- derived neurotrophic factor (BDNF); and neurotrophins 3 and 4 (NT-3, NT-4), were established using commercial ELISAs (Promega Wl). Specific protein concentrations were expressed relative to total protein concentration. In all cases n = 14.

EXAMPLE 13 Reverse transcription-PCR of the neurotrophins and their receptors

Brains were homogenised in Solution D (4M guanidinium thiocyanate, 25mM sodium citrate, pH7.0; 0.5% sarcosyl, 0.1M 2- mercaptoethanol). Total RNA was isolated as described by Chomczynski and Sacchi (1986). Superscript Choice System (Life Technologies) was used for reverse transcription of 5 μg of total RNA from 10 control animals and 10 vitamin D depleted animals. PCR conditions were optimised by varying MgCI₂ concentration and cycle number to determine linear amplification ranges for each primer pair. PCR products were identified by size and confirmed by DNA sequencing. Statistics All comparisons were conducted blind to treatment. All statistical comparisons between those animals raised under vitamin D normal and deplete conditions were by MANOVA with post-hoc significance assessed by either parametric or nonparametric Mann U Whittney test. Where only a single variable was compared between two groups, significance was assessed by an unpaired t test.

EXAMPLE 14

Testing a Potential Drug for Treating a Disorder A procedure for testing for a potential drug for treating a disorder is exemplified by schizophrenia. However, a similar procedure may be used to test another disorder as would be understood by a person skilled in the art.

An animal model displaying schizophrenic behavior may be used to screen a drug for potential anti-schizophrenic activity. A potential anti-schizophrenic drug is administered to the animal model in various amounts and an effect upon the schizophrenic behavior is monitored. The method of administration depends upon the drug being tested and can include oral, parenteral, transdermal or rectal administration. An effective drug is one that reduces schizophrenic behavior to a degree that approaches a behavior of a control or sham animal, e.g. the behavior changes towards normal behavior. For example, one could use a drug (for example, haloperidol, clozapine, or other currently available antipsychotic drugs) and measure the drug's effect on pre-pulse inhibition or any other behavior as described herein. These could include animal behaviours related to negative symptoms such as reduced social interaction, withdrawal, avoidance behaviour, reduced grooming, reduced sexual activity, other altered social behaviours and hyperlocomotion. In addition, the model may be used to examine various measures related to memory (maze tasks, latent inhibition), information processing (PPI, other startle paradigms) and other behaviours thought to be correlated with schizophrenia. If the abnormal behavior of the animal is found to decrease, i.e., be reduced, the drug is effective. An example of a method for screening a potential drug for treating schizophrenia is described in US Patent No.5,549,884, incorporated herein by reference. In this US patent, a group of surgically brain lesioned rats were treated for 3 weeks with either drug delivery vehicle or haloperidol to assess an effect of neuroleptic treatment on hyperlocomotion. The duration of treatment was chosen to approximate the subchronic duration of haloperidol administration associated with clinical response in patients with schizophrenia (Pickar, et al., Schizophr. Bull. 14:255-268 (1988)). At postnatal day 35, neonatally operated rats were randomly assigned to four groups: SHAM/VEH, LESION/VEH, SHAM/HAL, LESION/HAL, N=7/group. The first two groups were treated once daily with vehicle (VEH, water with a drop of TweenδO.RTM., an emulsifier and dispersing agent, adjusted to pH 5.6) given i.p. for 3 weeks, while the other two groups were injected with haloperidol (HAL, 0.4 mg/kg, suspended in VEH) over the same period of time. Half an hour after the last dose of haloperidol (PD-56), the rats were placed in photocell monitors and their locomotion activity was assessed for 1 h.

Swerdlow, 1994 describes a method for testing in an animal a response to a particular antipsychotic drug by testing a PPI response in an animal having a schizophrenia-like phenotype before and after administration of a potential antipsychotic drug.

Second set of experiments

Methods Animals and Housing

One hundred and nineteen male and female Sprague-dawley rats were used. Rats were maintained in a single holding room on a 12-h light-dark cycle (lights on at 0600 h). Lighting was provideD by incandescent bulbs giving a light level of 200-500 lux depending on the position ofthe cage within the rack. Housing was in plastic cages on racks holding 18 cages.

Animals were housed in same-sex litter mate groups

Experimental groups.

Animals were bred and reared at Herston Animal facility and assigned randomly to one of four experimental groups. Control dams were fed a normal diet (Dyets Inc CA USA) and vitamin D deficient dams were given a similar diet which was deficient in vitamin D from 4 weeks of age.

Dams were mated at 10 weeks of age and the timing of mating recorded. At birth the litters were reduced to three males and three females. In the first group the dams and offspring ate a control diet. In the second group vitamin D deficient dams were put onto a control diet at birth (Day 0) and the pups were placed on the control diet at weaning. In the third group pups born to vitamin D deficient dams were put onto the control diet at weaning (on postnatal day 21 (Day 21). The pups in the fourth group were weaned from vitamin D deficient dams and remained on a vitamin D deficient diet until the end ofthe experiment. The rat offspring (but not the dams) received calcium supplementation in the water (2 mM) after weaning. The animals were transferred to the behavioural testing facility and allowed three days to acclimatize. Most of the animals were then housed individually (96 in total) three males and three females from each group were housed in groups of three (23 in total) General Procedure

Behavioural testing started when the rats were 70 days old. On day 1 all animals were tested on the holeboard test for 7 min. On day two the rats were tested on the elevated plus maze for 7 min. On the third day the rats were tested in the social interaction test for 7 min followed by the prepulse inhibition of the acoustic startle response. On the fourth and fifth day the rats were tested in the forced swim test. All testing took place under quite conditions and dim white light between 0700 and 1500. The testing order was randomized for group and sex type. Statistical Analysis

A multivariate ANOVA to analyse the main factors Sex (Male and Female) and Group (Control, Day 0, Day 21, Deplete all) was used.

Where appropriate, repeated measures ANOVA was used. Post-hoc comparisons were made with Dunnetts's t-test. P < 0.05 was used to indicate significance. Example 15

Anatomy

The pups development was measured in terms of ear and eye opening and incisor protrusion. Weight and body length (base of tail to tip of snout) at week 10 was also measured. At the end of the experiment (11 weeks of age) the rats were killed by decapitation and the brains removed for further analysis. The brains were weighed and their size estimated. A terminal blood sample was taken for analysis.

There were significant effects of Group on body weight {F_3,HQ =

41.08, P < 0.001) and body length {F_3ι119 = 13.53, P < 0.001). Rats in the Deplete all group were lighter and shorter than control rats (P < 0.001), whereas Day 0 and Day 21 were the same size as control rats (P > 0.05).

Males were significantly heavier (Fι_tng = 685.10, P< 0.001) and significantly longer than females (F^₉ = 90.80, P< 0.001). The interaction between the main factors Sex and Group were significant for body weight

= 4.80, P < 0.005) and body length {F₃, = 2.89, P < 0.05). Deplete all males were significantly lighter and shorter than control males and Deplete all females were significantly lighter and shorter than control females (P < 0.05).

A sub sample of brains were taken at the end ofthe experiment (n = 9-12 for each sex per group), weighed and an estimate of brain width and brain length made. There were significant effects of the main factor Group on brain weight {F_3ι82 - 3.29, P< 0.05) and brain width {F_3ι82 = 3.67, P < 0.05) but not on brain length {F_3t82 = 2.45, P = 0.07). Post-hoc analysis indicated that Deplete all rats had lighter brains than control rats, whereas Day 0 rats had wider brains than Deplete all rats. However, when corrected for body weight Deplete all rats had significantly larger brains (weight, width and length) per g of body weight than rats in the remaining groups (control, Day 0 and Day 21 ; F_3ι82 = 22.19, P < 0.001). There was a significant effect of Sex on brain size. Males had larger {F_1>82 = 71.86, P < 0.001), longer {F_1t82 = 11 -06, P < 0.005) and wider {Fi,₈2 = 32.49, P < 0.001) brains than females. When corrected for body weight (brain weight/body weight) females had significantly larger brains per g of body weight than males {F_1>82 = 226.81 , P < 0.001). EXAMPLE 16

Holeboard test

A holeboard test was modified from that described by File and Wardill, 1975, Psychopharmacologia, 44 47, incorporated herein by reference. Briefly, the holedboard comprised a square open field (60 x 60 x 30 cm) made of opaque grey acrylic. A raised floor insert (5 cm above the floor) with four holes (5 cm diameter) were situated 10 cm in from each ofthe corners. The light level at the center of the floor was 100 lux. Behaviour was recorded from a centrally placed video camera. Distance traveled

FIG. 21 A shows a significant effect ofthe main factor Group on the distance traveled {F_3, = 16.89, P < 0.001). Post-hoc analysis with Dunnett's t-test showed that day 0 rats traveled significantly further than control rats (P < 0.001 ). There was a significant effect of the main factor Sex on the distance traveled {F₃,₁₁₉ = 7.27, P < 0.05); females (1969 ± 71 cm) traveled further than males (1784 ± 71 cm). The time spent in the centre of the holeboard did not differ between groups {F_3ιn₉ = 0.65, P- 0.58). There was no significant interaction between the factors Group and Sex on the distance traveled (F₃,m = 1-07, P = 0.37).

There were no significant effects of the factor Group on the frequency of grooming {F₃,n₉ = 1.70, P = 0.17), rearing {F₃,n9 - 1 -12, P = 0.34), number of faecal boli {F_3ιng = 2.09, P= 0.11) or pools of urine {F_3ιn₉ = 0.63, P = 0.60). Females had significantly higher scores for grooming {F_3ιn₉ = 9.34, P < 0.005) and rear {F_3ι119 = 4.95, P < 0.05) than males, but there was no effect of the main factor Sex on the number of faecal boli {F_3ι119 = 2.23, P = 0.14) or pools of urine {F_3ι119 = 2.23, P = 0.14).

There were no significant interactions between the main factors Sex and Group on the scores for groom, rear, defaecate or urinate (P > 0.05).

Frequency of head dips

As shown in FIG.21 B, there were no significant main effects of the factors Sex {F_1tn₉ = 1.80, P= 0.18) or Group {F_3>119 = 2.49, P= 0.06) on the frequency of head dips. The interaction of Sex and Group did not reach significance {F_3ι119 = 0.93, P = 0.43).

EXAMPLE 17 Elevated plus maze An elevated plus maze was a modification of a design by

Pellow et al, 1985, J Neurosci Methods 14 149, incorporated herein by reference. It comprises two open arms (50 x 7 x 1 cm) and two closed arms (50 x 7 x 30 cm) that extended from a central platform (7 x 7 cm). Like arms opposed each other across the central platform. The maze was constructed from opaque grey acrylic and was elevated on a metal stand 60 cm above a speckled grey linoleum floor. The light level at the central platform was 100 lux. An amount of time spent on the open and closed arms ofthe maze was measured. The percent open arm time was calculated as (time spent on open arm / time spent on close arm *100). A number of arm changes was also measured.

Time spent in open and closed arms

FIG. 22A shows no significant effect of Group on the amount of time spent in the open (F₃,n₉ = 0.89, P = 0.45) or the closed arm (F_{3|1 9} = 1.84, P = 0.14) or on the percent open arm time (F_3,n₉ = 1.18, P < 0.32). There were no significant effects of the main factor Sex (Fι_,n₉ < 0.80, P > 0.38; for each comparison) nor on the interaction of the factors Sex and Group (F₃,ιi9 < 1.12, P > 0.32; for each parameter) on the number of arm changes, the time spent in the open arm, the time spent in the closed arm or the percent open arm time. Number of arm changes

FIG.22B shows a significant effect ofthe main factor group on the number of arm changes (F₃,n9 = 6.03, P < 0.005). Day 0 rats made significantly more arm changes than rats in the other groups (control, Day 21 or Deplete all, P < 0.05).

EXAMLE 18 Social interaction

A social interaction test was performed in a circular arena (120 cm diameter x 30 cm high walls) made of opaque grey acrylic; the illuminance at the center of the floor was 100 lux. A camera was mounted above the arena and attached to a video monitor for observing behaviour.

The test consisted of placing a test rat and a younger rat (4-7 weeks of age) of the same sex at opposite sides of the test arena. A scorer, blind to treatment, recorded the total time spent investigating (sniffing and grooming) by each pair of rats in a 7 min test. Using this procedure no aggressive behaviours were observed. In addition, grooming and rearing were scored.

FIG. 23 shows a significant main effect of group on the time spent in social interaction {F_3ιn₉ = 2.90, P < 0.05). Post-hoc comparisons indicated that "Deplete all" rats spent less time in social interaction than controls (P < 0.05). There was no difference in the scores from Day 0 and control rats (P> 0.05), or from and Day 21 and control rats (P> 0.05). There was a main effect of the factor Sex on social interaction; males spent significantly longer engaged in social interaction than females Fι_tn₉ = 16.04, P < 0.001 ; mean duration (s) ± SEM; males: 85.87 ± 4.26, n = 60; females: 64.93 ± 3.28, n = 59). The interaction of the factors Sex and Group was not significant (F_3ι11g = 1.10, P = 0.35) EXAMPLE 19

Acoustic startle response

FIG. 24 shows that there was no significant main effect of the factor Group on the acoustic startle response (Average amplitude: F_3,n₉ = 0.41 , P = 0.74; Peak amplitude: F_3ιu₉ = 0.36, P = 0.78; Latency to peak: F_3iiig = 0.34, P = 0.79). There was a significant effect of the pulse intensity on the acoustic startle response (Average amplitude: F₅ = 210.44, P < 0.001 ; Peak amplitude: F_S = 184.32, P < 0.001 ; Latency to peak: F_5ι7 = 107.40, P < 0.001).

Increasing the pulse intensity resulted in a reduction in startle latency and an increase in both peak amplitude and average amplitude of the startle response. There was a significant main effect ofthe factor Sex on the average amplitude of the acoustic startle response Fι_,n - 5.79, P < 0.05), in which males had higher scores than females, but not on the peak amplitude (Fι_ι119 = 3.50, P = 0.06) or the latency to peak Fι, = 2.38, P = 0.13). There was a significant interaction between the factors Sex and the acoustic startle response (Average amplitude: Fs u = 4.22, P< 0.005; Peak amplitude: F_{5 14} = 2.45, P< 0.05; Latency to peak: F_5t714= 107.40, P= 0.06). Males had significantly greater scores than females for the peak and average amplitude at 110 and 120 dB (P < 0.05). There were no other significant interactions of the main factors (P > 0.05)

EXAMPLE 20 Prepulse inhibition of the acoustic startle response PPI was tested in four startle chambers (SR_Lab, San Diego

Instruments, San Diego, CA) comprising a clear plexiglass cylinder (8 cm diameter, 20 cm length) mounted on a plexiglass platform in a ventilated and sound-attenuated enclosure. A speaker was located 24 cm above the plexiglass chamber and a piezoelectric accelerometer was mounted below the plexiglass frame. A personal computer delivered sound bursts through the speaker and recorded motion in the stabilimeter. The startle response over 200 ms after stimulus onset was recorded. A startle session started with a 5 min acclimation period with a 70 dB background noise. The computer recorded activity for 200 ms at 20 sec intervals during the 5 min acclimation to obtain a background level of activity. After the acclimation period 5 startle pulses (110 dB) of broad band burst were presented for 40 ms with an inter stimulus interval (ITI) of 20 sec to test for basal startle responsiveness. Next five blocks of 15 trials were presented consisting of six different trial types of pulse alone trial (70, 80, 90, 100, 110 and 120 dB) and nine different trial types of prepulse and pulse trial. The pulse was presented for 40 ms and the prepulse of 20 ms broad band burst. The prepulse had an intensity of 74, 78 or 86 dB and preceded the pulse by 8, 32 or 256 ms. Trial types were presented in pseudorandom order with an ITI of 10-20 s (average 15 s).

The startle response (latency, peak amplitude and average amplitude) was recorded for each trial type and averaged for each trial type. The percentage PPI was calculated as [100 - (100 x startle amplitude on prepulse trial)/(startle amplitude on pulse alone trial)].

The percentage PPI data were calculated for average startle amplitude and peak startle amplitude (Vmax). As these data were highly correlated (Pearson correlation rs > 0.88, P < 0.0001 , n = 119, for each prepulse trial) only the percentage PPI scores calculated from the peak startle amplitude data are shown in FIG. 25. There was a significant main effect of Group on the percentage PPI scores {F_3ιn₉ = 4.91 , P < 0.005; repeated measures ANOVA).

Post-hoc comparisons with a Dunnett t test (2-sided) indicated that the Controls had lower percent PPI scores than those from Day 0 (P < 0.05) and Day 21 (P < 0.005), but not Deplete all rats (P = 0.22). There was no significant main effect of Sex on the percent PPI scores {F ,ng = 0.61 , P = 0.44). There was a significant main effect of the prepulse stimulus on the percent PPI scores {F_8j952 = 110.39, P < 0.0001). Increasing the prepulse intensity at a constant prepulse/pulse interval (8, 32 or 256 ms) resulted in significantly higher percent PPI scores. The interactions of the main effects were not significant (PPI x Sex: F_8>952 = 1.74, P = 0.09; PPI x Group: F_8ι952 = 1.28, P = 0.16; Sex x Group: F_3,n₉ = 1.23, P = 0.30; PPI x Sex x Group:

Latency to startle during the prepulse trials

There were no significant main effects of the factors Group {F₃,ng = 0.50, P = 0.68) or Sex {Fι,ng = 0.05, P = 0.83) on the latency to startle during the prepulse trials. There was a significant effect of the prepulse stimulus on the latency to startle {F_8t952 = 21.00, P < 0.001 ).

EXAMPLE 21 Forced swim test

A forced swim test was based on methods commonly used in the art, for example those described by Porsolt et al, 1977, Nature 266730, incorporated herein by reference. A white opaque cylinder (30 x 45 cm) was filled to a predetermined level with water (30°C) depending on a length of a test rat. Rats were measured from tip ofthe snout to the base ofthe tail and the water depth calculated by multiplying this value by 1.25. Thus, a 20 cm long rat was placed in water to a depth of 25 cm. The rats were tested over two days. On the first day they were placed in the water and 10 min later removed and dried with towels. The rat was placed in a cage with paper towel for 20 min and then returned to its home cage. Twenty-four hours later they were returned to the water for 5 min.

The behaviour was scored from a video monitor by a scorer blind to treatment. We evaluated the time spent in the following categories; immobile - no movement of the paws and/or floating (small movements of the paws to maintain the head above water); mobile -sufficient movement of the paws to facilitate movement; scrabbling - active movement ofthe paws on the side of the container; and diving - dipping the head below horizontal beneath the water. Escape behaviours were defined as the combined scores for scrabbling and diving. Duration of time immobile FIG.26A shown no significant main effects of the factor Group on the duration of time immobile {F₃,n₉ = 1.08, P= 0.36) or on the duration of time performing escape behaviours {F_3ιu₉ = 0.37, P = 0.78). Females were immobile for significantly longer than males Fι,n - 45.22, P < 0.001 ; mean duration (s) ± SEM; males: 38.03 ± 4.23, n = 60; females: 120.49 ± 11.53, n = 59), whereas, overall, males spent longer performing escape behaviours than females {Fι,ug = 9.07, P < 0.005; mean duration (s) ± SEM; males: 60.65 ± 6.80, n = 60; females: 35.73 ± 5.02, n = 59). The interaction of the factors Sex and Group was significant for escape behaviours {F_3ιng = 2.71 , P < 0.05) but not for the time spent Immobile {F_3ιu₉ = 1.18, P= 0.32). Post-hoc analysis with Dunnetts t-test showed that Male Day 21 rats spent significantly longer performing escape behaviours than female Day 21 rats. Duration of escape

There was no significant difference between males and females for the time spent trying to escape in the remaining groups (control, Day 0 or Deplete all), see FIG. 26B.

It is understood that the invention described in detail herein is susceptible to modification and variation, such that embodiments other than those described herein are contemplated which nevertheless falls within the broad scope of the invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.

Table 1

Source num. df den. df F V

Group 5 142 10.15 001

Sex 1 142 9.09 .003

Trial⁰ 2 284 56.70 <.001

Age 1 93 35.02 <.001

Group x Sex 5 142 0.53 0.75

Group x Trial 10 284 0.59 .82

Group x Age 4° 93 5.79 <.001

Sex x Trial 2 284 0.49 .61

Sex x Age 1 93 2.70 .10

Trial x Age 2 186 1.70 .19

Group x Sex x Trial 10 284 0.57 .83

Group x Sex x Age 4 93 2.05 .09

Group x Trial x Age 8 196 0.28 .97

Sex x Trial x Age 2 186 0.41 .66

Group x Sex x Trial x Age 8 186 0.39 .92

Post-hoc analysis of Group x Week means

Groups (excluding No Nit D) x Age 3 93 0.39 .76

No Nit D x Age 1 93 2.90 .09

Group Within Week 5 4 93 1.49 .21

Group Within Week 10 5° 93 11.51 <.001^d

No Nit D v. other cohorts (Week 10) 1 93 56.77 001^d

Other Group Within Week 10 4 93 0.56 .69

Cohorts: Day 0, Day 30, Controls, No D, Normal, Adult.

Trials: PP-P70 PP-P75 PP-P80 ^c ^^"reflect the availability of 5 cohorts at week 5, and 6 at week 10. ^d After Bonferroni adjustment for the post-hoc comparison. Table 2

GROUP

Late Norm Con DO D30 NoD

Males 5 Weeks 114 + 5 126 + 7 117 + 4 105 + 6 112 + 8

10 Weeks 457 + 17 331 + 9 334 + 16 309 + 9 300 + 11 218 + 21

Females 5 Weeks 108 + 4 100 + 7 102 + 5 94 + 5 99 + 7

10 Weeks 297 + 11 231 + 14 224 + 9 215 + 5 205 + 7 152 + 8

Table 3

Morphological features (mean ± SE)

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Claims

1. A method for producing a non-human animal characterized by a modified behavior when compared with a normal animal, the method including the step of at least reducing an amount of vitamin D, vitamin D precursor or vitamin D-like compound of the mother of the non-human animal.

2. The method of claim 1 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound is reduced in the mother at least prior to weaning of the non-human animal.

3. The method of claim 2 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound is reduced in the mother prior to birth of the non-human animal.

4. The method of claim 3 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound is reduced in the mother prior to pregnancy.

5. The method of claim 4 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound is reduced in the mother at least two weeks prior to pregnancy.

6. The method of claim 5 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound is reduced in the mother at least four weeks prior to pregnancy.

7. The method of claim 1 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound of the mother is measured as a 25 Hydroxy vitamin D plasma concentration of less than 15 ng/ml. 8. The method of claim 7 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound of the mother is measured as a 25 Hydroxy vitamin D plasma concentration of less than 5 ng/ml. 9. The method of claim 1 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound ofthe mother is reduced by feeding said mother a diet deficient in an amount of vitamin D, vitamin D precursor or vitamin D-like compound.

10. The method of claim 1 wherein the non-human animal born from the mother has at least a reduced amount of vitamin D, vitamin D precursor or vitamin D-like compound.

11. The method of claim 10 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound of the non-human animal born from the mother is reduced at least during early development. 12. The method of claim 11 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound of the non-human animal born from the mother is reduced at least from birth to weaning.

13. The method of claim 11 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound of the non-human animal born from the mother is reduced at least from birth to adulthood.

14. The method of claim 10 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound of the non-human animal born from the mother is measured as a plasma concentration of 25 Hydroxy vitamin D less than 30 ng/ml.

15. The method of claim 14 wherein the plasma concentration of 25 Hydroxy vitamin D is less than 5 ng/ml.

16. The method of claim 10 wherein the amount of vitamin D, vitamin D precursor or vitamin D-like compound of the non-human animal born from the mother is reduced by feeding the animal a diet comprising a deficient amount of vitamin D, vitamin D precursor or vitamin D-like compound.

17. The method of claim 9 or claim 16 wherein the diet comprises no detectable vitamin D, vitamin D precursor or vitamin D-like compound.

18. The method of claim 1 or claim 10 wherein the modified behavior includes hyperlocomotion, decreased social interaction and/or a change in prepulse inhibition.

19. The method of claim 1 or claim 10 wherein the animal born from the mother is a means for assessing a behavioral disorder.

20. The method of claim 19 wherein the behavioral disorder is a neuro-psychiatric disorder.

21. The method of claim 20 wherein the neuro-psychiatric disorder is characterized by hyperlocomotion, decreased social interaction and/or a change in pre-pulse inhibition when compared with a normal animal.

22. The method of claim 20 wherein the neuro-psychiatric disorder is selected from the group consisting of: schizophrenia, multiple sclerosis, anxiety, autism, Alzheimer's disease, Parkinson's disease, depression, mania, attention deficit/hyperactivity disorder and dementia.

23. The method of claim 20 wherein the neuro-psychiatric disorder is characterised by increased dopaminergic tone.

24. The method of claim 20 wherein the neuro-psychiatric disorder is schizophrenia.

25. The method of claim 1 or claim 10 wherein the non-human animal is further characterized by a modified morphology when compared with a normal animal.

26. The method of claim 1 or claim 10 wherein the mother and animal born from the mother are not exposed to ultra violet B (UVB).

27. The method of claim 26 wherein the mother and animal born from the mother are only exposed to incandescent light.

28. The method of claim 1 or claim 10 wherein the non-human animal is a mammal. 29. The method of claim 28 wherein the mammal is a rat or mouse.

30. A non-human animal produced according to the method of claim 1 or claim 10.

31. A non-human animal characterized by a modified behavior when compared with a normal non-human animal, said non-human animal being a progeny of a mother having at least a reduced amount of vitamin D, vitamin D precursor or vitamin D-like compound at least prior to giving birth to said non-human animal.

32. The non-human animal of claim 31 wherein the non-human animal has at least a reduced amount of vitamin D, vitamin D precursor or vitamin D-like compound.

33. The non-human animal of claim 31 or claim 32 wherein the reduced amount of vitamin D, vitamin D precursor or vitamin D-like compound is a result of a diet deficient in vitamin D, vitamin D precursor or vitamin D-like compound.

34. The non-human animal of claim 31 or claim 32 wherein the modified behavior is characteristic of a disorder.

35. The non-human animal of claim 34 wherein the disorder is a neuro-psychiatric disorder.

36. The non-human animal of claim 35 wherein the disorder is selected from the group consisting of: schizophrenia, multiple sclerosis, anxiety, autism, Alzheimer's disease, Parkinson's disease, depression, mania, attention deficit/hyperactivity disorder and dementia. 37. The non-human animal of claim 36 wherein the neuro- psychiatric disorder is characterised by increased dopaminergic tone.

38. The non-human animal of claim 31 or claim 32 wherein the neuro-psychiatric disorder is schizophrenia.

39. The non-human animal of claim 31 or claim 32 wherein the non-human animal is a mammal.

40. The non-human animal of claim 39 wherein the mammal is a rat or mouse.

41. A method for identifying a behavior modifying molecule including the steps of:

(i) administering at least one candidate molecule to a non- human animal produced by the method of claim 1 , 10, 31 or 32; and (ii) assessing the animal for a behavioral change in response to said candidate molecule(s) to determine if there is a change from the modified behavior, wherein a change from the modified behavior indicates said candidate molecule(s) is a behavior modifying molecule. 42. The method of claim 41 wherein the modified behavior is behavior is selected from the group consisting of: hyperlocomotion, decreased social interaction and a change in pre-pulse inhibition when compared with a normal animal.

43. The method of claim 41 wherein the modified behaviour is characteristic of a behavioural disorder.

44. The method of claim 43 wherein said behaviour disorder is a neuro-psychiatric disorder.

45. The method of claim 44 wherein the neuro-psychiatric disorder is a disorder characterized by increased dopaminergic tone.

46. The method of claim 44 wherein the neuro-psychiatric disorder is selected from the group consisting of: schizophrenia, multiple sclerosis, anxiety, autism, Alzheimer's disease, Parkinson's disease, depression, mania, attention deficit/hyperactivity disorder and dementia.

7. The method of claim 46 wherein said neuro-psychiatric disorder schizophrenia.