460. DIETARY CUPRIZONE AND GESTATIONAL THC EXPOSURE BOTH INDUCE LOCALIZED DEPLETION OF BRAIN COPPER

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Abstract Background Neurobehavioural abnormalities associated with neurodevelopmental disorders are associated with a complex interplay of genetic and environmental factors. Models of these conditions commonly involve early-life environmental insult, such as chemical or drug exposure, or inflammatory events. Additionally, demyelination pathology associated with psychiatric illnesses such as schizophrenia may be modelled with dietary cuprizone administration. Despite cuprizone toxicity as a common model of demyelination, its modes of action are not known, though copper deficiency is suggested to underlie the mechanism. In the case of THC, the effects of THC exposure on brain Cu levels during early life in rats is completely unknown, although early life THC exposure is reported to impact oligodendrocyte proliferation and maturation. Aims & Objectives Our aim was to directly visualise Cu distribution at micron spatial resolution with nM detection limits to determine if brain-copper levels are disturbed in two distinct models associated with neurobehavioural abnormalities. Method For the cuprizone experiments, C57BL/6 mice (male, 8-weeks age) were fed 0.2% w/w cuprizone-containing diet or control diet for 1, 3 or 6 weeks, or 6-weeks cuprizone plus 4-weeks control diet for remyelination (n=7/group). Brain tissue was collected at the end of the designated exposure period. For the gestational THC experiments, female Sprague-Dawley rats were bred in-house and administered daily THC (3mg/kg; n=5) or vehicle (1:1:18 Kolliphor, ethanol, saline; n=3) intraperitoneally from gestational day 6-20. Following natural parturition, offspring were left undisturbed with their mother until weaning at postnatal day 21. Offspring brain tissue collection occurred at adulthood, postnatal day >90 (n=5/group). For both experiments: Immediately following humane euthanasia, tissue was rapidly dissected and frozen in liquid nitrogen-cooled isopentane without perfusion or fixation. Cryosections were air-dried onto X-ray transparent substrate for elemental mapping with X-ray fluorescence microscopy (XFM) at ANSTO-Australian Synchrotron (cuprizone) or Canadian Light Source (THC). Results For the cuprizone experiments, 2-way ANOVA of myelin density (luxol fast blue) revealed pronounced cuprizone-induced demyelination in lateral corpus callosum, medial corpus callosum, lateral striatum white matter, and medial striatum white matter. One-way ANOVA revealed significant localised reduction in copper in lateral cortex dorsal to the corpus callosum, and striatum, but not medial regions. For the gestational THC experiments, unpaired two-tailed t-test revealed significantly reduced copper in the perimeter of the corpus callosum. Discussion & Conclusions This is the first-time brain-Cu has been directly visualised in the cuprizone demyelination model and in gestational THC exposure. Notably, both environmental insults resulted in localized brain-Cu deficiency at the perimeter of the corpus callosum. Brain-copper dysregulation may occur as a common pathological feature across diverse disease models. These findings may lead to new insights into glial biology, demyelinating diseases, and neurodevelopmental disorders.

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