Hyperactivity and alterations in iron homeostasis in mu opioid receptor knockout mice: possible implications for restless legs syndrome/Willis-Ekbom disease

Introduction Restless legs syndrome (RLS), also known as Willis-Ekbom disease, is a neurological disorder that is manifested generally at rest by an urge to move and is often accompanied by unpleasant sensations in the legs. Studies have demonstrated that the degree of opioid receptor binding is inversely proportional to the severity of RLS symptoms and there is a decrease in thalamic beta-endorphin and met- enkephalin, which are endogenous opioid peptides, in RLS patients. Furthermore, μ - opioid receptor agonists have been used to treat RLS patients. In this study we examined the relevance of the mu opioid receptor to RLS. Materials and methods To test the hypothesis that the μ -opioid receptor plays a role in RLS pathophysiology we have taken advantage of a previously generated μ -opioid receptor knockout (KO) mouse. We analyzed the KO mice for RLS-like phenotypes, including spontaneous activity levels, voluntary wheel running levels during the day and night, and sleep structure. Additionally, we measured the levels of neurotransmitters and their metabolites in the striatum using high performance liquid chromatography. Lastly, we examined the KO mice and mice treated with a potent irreversible antagonist for the μ -opioid receptor for alterations in iron homeostasis in the brain, liver, spleen, and serum using atomic absorption spectroscopy, colorimetric assays, and Western blot analysis. Results We have found that mice deficient for the μ -opioid receptor have increased wheel running activity during the rest phase, parallel to human RLS. Furthermore, we have found that loss of the μ -opioid receptor or pharmacological blockade of the mu opioid receptor using an antagonist leads to alterations in iron homeostasis in the periphery, in particular in the serum and spleen. However, we did not observe any alterations in the dopaminergic, serotonergic, or iron systems in the striatum of the μ -opioid receptor KO mice. Conclusion Taken together, while the μ -opioid receptor knockout mice do not match human RLS perfectly, they do have sleep period hyperactivity as expected of an animal model of RLS. Furthermore, the data suggest an interesting role of the mu opioid receptor in iron homeostasis that will need to be investigated in detail in future studies. Acknowledgements We thank Chad C. Cheetham, Atbin Doroodchi, Miki Jinno, Ning Peng, and J. Michael Wyss for their technical assistance and stimulating discussions. This work was supported by the National Institutes of Health (grants NS37409, NS47466, NS47692, NS54246, NS57098, NS65273, NS72872 and NS74423) and startup funds from the Departments of Neurology at UAB and UF.