The sulphydryl reagent, N-ethylmaleimide, disrupts sleep and blocks A1 adenosine receptor-mediated inhibition of intracellular calcium signaling in the in vitro ventromedial preoptic nucleus

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To explore the neuronal signaling mechanisms underlying sleep regulation in the rat, the present study examined continuous intra-third ventricle infusion of N-ethylmaleimide (NEM), a sulphydryl reagent that inhibits G i/o protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01–10 μmol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 μmol/10 h) dramatically inhibited day-time sleep (−57% for non-rapid eye movement sleep and −89% for rapid eye movement sleep) with a compensatory increase of sleep during the subsequent night-time (+33% for non-rapid eye movement sleep and +259% for rapid eye movement sleep). The day-time brain temperature was also increased by NEM, demonstrating effects of NEM on both sleep and body temperature levels. Immunostaining of the rat hypothalamus with a monoclonal antibody against the A1 adenosine receptor (A1R) was used to explore the distribution of a sleep-related G i/o protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca 2+ imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N 6-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca 2+ oscillations and high potassium (80 mM)-induced Ca 2+ flux in the ventromedial preoptic nucleus, while NEM (100–300 μM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca 2+ flux. From these results we suggest that NEM-sensitive G protein-coupled receptor(s) may play an important role in the regulation of sleep and body temperature in the rat and one possible mechanism is an A1R-mediated regulation of intracellular Ca 2+ concentrations in the ventromedial preoptic nucleus.