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 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.