<p>We hypothesized that Restless Legs Syndrome (RLS) and the restlessness of opioid withdrawal share common neurobiological mechanisms based on the efficacy of μ-opioid receptor (MOR) agonists in RLS and the common RLS-like phenotype of patients with opioid withdrawal. We also hypothesized this involves an increased sensitivity of the striatal striosomal neurons that co-express MORs and dopamine D₁ receptors (D₁Rs) and release GABA in the internal segment of the globus pallidus (GPi). This hypothesis was tested in mice with diet-induced brain iron deficiency (BID), a rodent model of RLS. Fiber-photometry experiments were performed in mice with BID using a viral GABA biosensor injected in the entopeduncular nucleus (EPN), the GPi equivalent in rodents. EPN GABA release was measured after the systemic administration of the D₁R agonist SKF81297 and the MOR agonist methadone. Locomotor activation and striatal mRNA expression of D₁Rs, MORs and adenosine A₁ receptors (A₁Rs) were also analyzed. A minimal locomotor-activating dose of SKF81297 induced a significant EPN GABA release in mice with BID but not controls, while a maximal locomotor-activating dose of the MOR agonist methadone significantly reduced EPN GABA release in mice with BID after saline or SKF81297 administration. BID was associated with a significant reduction in the striatal expression of MORs and A₁Rs. The results indicate that BID induces an increased dopaminergic sensitivity of the opioid-responsive striatal–EPN pathway, which might represent a pivotal pathogenetic mechanism of the restlessness of RLS and opioid withdrawal.</p>