MECHANISMS OF SPONTANEOUS CALCIUM OSCILLATIONS AND ACTION-POTENTIALS IN IMMORTALIZED HYPOTHALAMIC (GT1-7) NEURONS

1. Individual immortalized gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons in semiconfluent cultures showed spontaneous oscillations in intracellular Ca2+ concentration ([Ca2-](i)) as measured by video fluorescence microscopy and fura-2. In parallel experiments, GT1-7 neurons also showed spontaneous bursts of action potentials that were recorded as action currents from intact cells. The bursts of action currents occurred in characteristic patterns, suggesting an underlying rhythmic oscillation in membrane potential. 2. Depolarization with increased extracellular K+ evoked a concentration-dependent increase in the Frequency of Ca2+ oscillations or a sustained plateau of increased [Ca2+](i) in GT1-7 neurons. Increased extracellular K- (30 mM) caused an initial increase in the frequency of action currents, after which they were reversibly abolished. 3. The Ca2- channel blockers Ni2- and nimodipine abolished Ca2+ oscillations, whereas nifedipine, gadolinium, omega-conotoxin and omega-agatoxin had no effect on Ca2+ oscillations. These results indicate that Ca2- oscillations are generated by influx of Ca2- through voltage-gated Ca2- channels that are not sensitive to nifedipine and are not N-type or P-type channels. 4. Thapsipargin caused a small, transient rise in baseline [Ca2+], but had no effect on Ca2- oscillations. Caffeine and ryanodine had no effect on baseline [Ca2-], or Ca2+ oscillations. These results indicate that the release of Ca2+ from inositol 1,4,5-triphosphate (IP3)-sensitive or caffeine-sensitive intracellular stores does not play a major role in Ca2+ oscillations in GT1-7 neurons. 5. Carbonyl-cyanide p-trifluoromerhoxyphenyl hydrazone caused an immediate increase in baseline [Ca2+](i) the frequency of Ca2+ oscillations, and the frequency of action potentials, which suggests that mitochondrial Ca2+ stores play a role in Ca2+ oscillations in GT1-7 neurons. 6. The K+ channel blockers quinine and Ba2+ evoked a concentration-dependent increase in the frequency of action potentials or a sustained plateau of increased [Ca2+](i). Tetraethylammonium increased the amplitude of Ca2+ oscillations by two- to threefold. The different effects of the different K+ channel blockers suggest specific roles for different types of K+ channels in the generation of Ca2+ oscillations. 7. Neither GnRH nor a GnRH antagonist had any effect on Ca2+ oscillations, indicating that Ca2+ oscillations were not influenced by release of GnRH. 8. We propose that intrinsic oscillations in membrane potential driven by K+ channels activate action potentials and an influx of Ca2+ through nimodipine-sensitive Ca2+ channels to generate Ca2- oscillations in GT1-7 cells. This intrinsic activity may underlie the spontaneous release of GnRH.