CHARACTERIZATION OF SPONTANEOUS AND N-METHYL-D-ASPARTATE-INDUCED CALCIUM RISE IN RAT CULTURED HYPOTHALAMIC NEURONS

The effect of N-methyl-D-aspartate (NMDA) on intracellular calcium concentration ([Ca2+](i)) was analyzed in cultured hypothalamic neurons using the Ca2+-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-H-5-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 Ca2+-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 mu M) 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 mu M), and ryanodine (10 mu M) 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 mu MTTX but at 1 mu M 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.