|Description||Restless Legs Syndrome (RLS) is a sensorimotor disorder that severely disrupts sleep. RLS patients regularly present with a condition known as Brain Iron Deficiency (BID), and BID is commonly associated with altered dopamine and adenosine neurotransmission in the striatum. Dopamine and adenosine can form receptor heteromers in the striatum, where the adenosine A1 receptor (A1R) modulates dopamine D1 receptor (D1R) function. However, there are no data on the impact of BID in the spinal cord, the ultimate sensorimotor circuitry involved in RLS. We here tested if a diet-induced brain iron-deficient animal model affects spinal cord excitability as reported in other RLS animal models, and we tested the responsiveness of this model to treatment with dopamine and adenosine receptor modulators.
Following previously established protocols, C57Bl/6 mice were separated upon weaning into male and female cohorts fed either control iron or iron-reduced diets. The BID diet did not induce an anemic phenotype. To assess spinal cord excitability, we measured thermal pain reflex withdrawal latencies (RWLs) using the Hargreaves system, starting at one-week post-diet exposure. The BID cohorts showed significantly lower RWLs than their respective CTRL cohorts, and these differences remained stable over time. We then tested the responsiveness of this model to dopamine receptor modulators Pramipexole (PPX, D3 receptor agonist, 0.5 mg/kg, i.p.) and SCH 39166 (Ecopipam, D1 receptor antagonist, 0.5 mg/kg + 1.0 mg/kg, i.p.), and adenosine receptor modulators caffeine (A1R/A2R, 50 mg/kg, i.p.) and N6-cyclpentyladenosine (CPA, A1R agonist, 1.0 mg/kg, i.p.). These data indicate that PPX did have significant effects on increasing RWLs but with strong locomotor side effects, SCH 39166 showed significant effects in increasing RWLs in the male BID cohort, but not female BID cohort, and while caffeine did not have significant effects on RWLs in CTRL cohorts, use of CPA led to a significant increase in RWLs in both male and female BID cohorts. Western blot analysis of D1R and A1R expression in the mouse spinal cord revealed an increase in D1R expression and opposing decrease in A1R expression. Finally, the use of proximity ligation assays (PLAs) revealed the presence of A1R-D1R heteromers within the mouse spinal cord, where motoneurons reside, and that these heteromers decrease in number or are no longer functional under BID conditions.
Together our data show that diet-induced iron-deficiency leads to a decrease in RWLs, and that use of dopamine and adenosine receptor modulators show significant effects in this model. RLS patients present with BID and are initially highly responsive to dopamine D3 receptor (D3R)-based treatment in the clinic. However, long-term treatment with these compounds can lead to unwanted long-term side effects. We here propose a hypothetical model of RLS and how BID conditions might affect dopamine and adenosine neurotransmission via A1R-D1R heteromers.||