1. Changes in intracellular Ca2+ ion concentrations ([Ca2+]i) during potassium-induced depolarizations were studied in cultured embryonic Xenopus spinal neurones using the Ca2+-sensitive dye Fura-2 and quantitative fluorescence microscopy. 2. Membrane voltages attained during exposure to bath solutions containing 3, 10, 20, 30, 40 and 50 mM-K+ were determined under current clamp. In 3 mM-K+-containing solution (normal saline), the resting potential was -65 mV. The threshold voltage required to observe a measurable rise in [Ca2+]i was -40 mV (external potassium concentration [K+]o = 20 mM). The depolarization-induced [Ca2+]i signal had two components: a non-relaxing component, and, at voltages positive to -40 mV, an additional transient component on the rising phase that decayed over tens of seconds. There was substantial variability in the magnitudes of resting and voltage-induced changes in [Ca2+]i, but [Ca2+]i responses were qualitatively consistent between neurones of similar ages. 3. External potassium (K(o)+)-induced increases in [Ca2]i were spatially non-homogeneous. The largest increases were seen in the nucleus, near the base of a major neurite, and in growth cones. Increases occurred more rapidly in neurites and growth cones than in somas. T-type and high-voltage-activated (HVA) channels appeared to be present in all cell regions. 4. Increases in [Ca2+]i evoked by 50 mM-K+ (depolarization to approximately -15 mV) were sensitive to treatments demonstrated to inhibit Ca2+ currents in these cells (T-type, HVA-relaxing and HVA-sustained), including Ni2+ (200-mu-M), Metenkephalin (17.5-mu-M), and omega-conotoxin (omega-CgTx; 5.5-mu-M). [Ca2+]i increases were reduced by caffeine (10 mM) and ryanodine (10-100-mu-M), agents that affect Ca2+ release from intracellular stores. 5. A sustained increase in [Ca2+]i observed at approximately -40 mV ([K+]o = 20 mM) was investigated in greater detail. Concentrations of Ni2+ sufficient to block T-type Ca2+ current slowed but did not block the rise in [Ca2+]i induced by 20 mM-K+. Met-enkephalin did not affect the [Ca2+]i response. omega-CgTx reduced the amplitude of the [Ca2+]i response, but did not eliminate the sustained component. Verapamil (100-mu-M), caffeine and ryanodine differentially reduced the sustained component as compared to the initial rising phase. These observations suggest that the rising phase was due to Ca2+ influx through T-type and other Ca2+ channels, and that the sustained phase was differentially sensitive to inhibition of internal Ca2+ release. 6. The data indicate that slow subthreshold depolarizations can cause long-lasting changes in [Ca2+]i, and that at subthreshold voltages [Ca2+]i is influenced by interactions between Ca2+ entry through Ni2+- and omega-CgTx-sensitive pathways (including T-type Ca2+ channels), and ryanodine-sensitive Ca2+ release from intracellular stores. Thus Ca2+-dependent developmental processes promoted by subthreshold depolarizations may be modulated by both Ca2+ entry and intracellular Ca2+ release.
