title: Characterization of spontaneous and N-methyl-D-aspartate-induced calcium rise in rat cultured hypothalamic neurons.
abstract: The effect of N-methyl-D-aspartate (NMDA) on intracellular calcium concentration ([Ca2+]i) was analyzed in cultured hypothalamic neurons using the Ca(2+)-sensitive fluorescent dye Fura-2. The resting [Ca2+]i in silent neurons ranged between 35 and 100 nM and regular spontaneous [Ca2+]i oscillations were observed in 37% of neurons. Such [Ca2+]i oscillations were blocked by tetrodotoxin (TTX--the sodium channel blocker), and reduced by the voltage-sensitive calcium channel blockers omega-conotoxin (omega-CTX-GVIA) (N-type) and nifedipine (L-type). NMDA increased [Ca2+]i transients and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d')cyclohepten-5,10-imine hydrogen] reduced them, in a dose-response manner. The amplitude of the NMDA-induced [Ca2+]i rise increased with increasing external Ca2+ concentrations, and was completely abolished in Ca(2+)-free medium. The role of intracellular calcium was tested by addition of intracellular Ca2+ mobilizers. In the presence or absence of external Ca2+, 2,5-di(tert-buty)-1,4-benzohydroquinone) (tBuBHQ) (25 microM) evoked a robust [Ca2+]i rise in NMDA-sensitive neurons. Preincubation (20 min) with tBuBHQ completely abolished the NMDA-induced [Ca2+]i response. Caffeine (10 mM), thapsigargin (25 microM), and ryanodine (10 microM) did not elicit any Ca2+ transients. Nifedipine and omega-CTX-GVIA did not modify NMDA-induced [Ca2+]i transients. NMDA-induced [Ca2+]i rise was not altered by 0.1 microM TTX but at 1 microM it was reduced by 20%. These data show that hypothalamic neurons in culture respond to NMDA in a dose-dependent manner by a rise in [Ca2+]i and that this response is mediated by NMDA receptor-gated channel. In addition, [Ca2+]i rise is dependent on the presence of extracellular Ca2+, and also seems to involve mobilization of Ca2+ from tBuBHQ-sensitive intracellular stores.
authors.affiliation: Laboratoire de Neurobiologie Endocrinologique, URA 1197-CNRS, Université Montpellier II, France.
authors.forename: G
authors.initials: G
authors.lastname: Dayanithi
authors.forename: F
authors.initials: F
authors.lastname: Rage
authors.forename: P
authors.initials: P
authors.lastname: Richard
authors.forename: L
authors.initials: L
authors.lastname: Tapia-Arancibia
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D016291:Dizocilpine Maleate / Q000494:pharmacology; D007031:Hypothalamus / Q000187:drug effects* / Q000196:embryology / Q000378:metabolism; D008978:Mollusk Venoms / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013268:Stimulation, Chemical; D013779:Tetrodotoxin / Q000494:pharmacology; D020866:omega-Conotoxin GVIA
chemical_list: D002121:Calcium Channel Blockers; D008978:Mollusk Venoms; D010455:Peptides; D013779:Tetrodotoxin; D016291:Dizocilpine Maleate; D020866:omega-Conotoxin GVIA; D002118:Calcium