Résumé
The effect of N-methyl-D-aspartate (NMDA) on intracellular calcium concentration ([Ca 2+ ] i ) was analyzed in cultured hypothalamic neurons using the Ca 2+ -sensitive fluorescent dye Fura-2. The resting [Ca 2+ ] i in silent neurons ranged between 35 and 100 nM and regular spontaneous [Ca 2+ ] i oscillations were observed in 37% of neurons. Such [Ca 2+ ] i oscillations were blocked by tetrodotoxin (TTX – the sodium channel blocker), and reduced by the voltage-sensitive calcium channel blockers ω-conotoxin (ω-CTX-GVIA) (N-type) and nifedipine (L-type). NMDA increased [Ca 2+ ] i transients and MK-801 [(+)-5-methyl-10,11-dihydro- 5 H-dibenzo(a,d’)cyclohepten-5,10-imine hydrogen] reduced them, in a dose-response manner. The amplitude of the NMDA-induced [Ca 2+ ] i rise increased with increasing external Ca 2+ concentrations, and was completely abolished in Ca 2+ -free medium. The role of intracellular calcium was tested by addition of intracellular Ca 2+ mobilizers. In the presence or absence of external Ca 2+ , 2,5-di(tert-buty)-1,4-benzohydroquinone) (tBuBHQ) (25 µM) evoked a robust [Ca 2+ ]i rise in NMDA-sensitive neurons. Preincubation (20 min) with tBuBHQ completely abolished the NMDA-induced [Ca 2+ ] i response. Caffeine (10 mM), thapsigargin (25 µM), and ryanodine (10 µM) did not elicit any Ca 2+ transients. Nifedipine and ω-CTX-GVIA did not modify NMDA-induced [Ca 2+ ] i transients. NMDA-induced [Ca 2+ ] i rise was not altered by 0.1 µM TTX but at 1 µM 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 [Ca 2+ ] i and that this response is mediated by NMDA receptor-gated channel. In addition, [Ca 2+ ] i rise is dependent on the presence of extracellular Ca 2+ , and also seems to involve mobilization of Ca 2+ from tBuBHQ-sensitive intracellular stores.