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

M. Ikeda, M. Sagara, Y. Sekino, T. Shirao, K. Honda, T. Yoshioka, C. N. Allen, S. Inoué

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

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 Gi/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 Gi/o protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca2+ imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N6-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca2+ oscillations and high potassium (80 mM)-induced Ca2+ 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 Ca2+ 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 Ca2+ concentrations in the ventromedial preoptic nucleus.

Original languageEnglish (US)
Pages (from-to)733-743
Number of pages11
JournalNeuroscience
Volume106
Issue number4
DOIs
StatePublished - Oct 31 2001

Keywords

  • Ca imaging
  • Fura-2
  • GTP-binding protein
  • Immunostaining
  • Intracerebroventricular infusion
  • Voltage-sensitive Ca channels

ASJC Scopus subject areas

  • Neuroscience(all)

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