C a 2+ Transients Are Not Required as Signals for Long-term Neurite Outgrowth f om Cultured Sympathetic Neurons

A method for clamping cytosolic free Ca 2+ ([Ca2+]~) in cultures of rat sympathetic neurons at or below resting levels for several days was devised to determine whether Ca 2+ signals are required for neurite outgrowth from neurons that depend on Nerve Growth Factor (NGF) for their growth and survival. To control [Ca2+]i, normal Ca 2+ influx was eliminated by titration of extracellular Ca 2+ with EGTA and reinstated through voltage-sensitive Ca 2+ channels. The rate of neurite outgrowth and the number of neurites thus became dependent on the extent of depolarization by KC1, and withdrawal of KC1 caused an immediate cessation of growth. Neurite outgrowth was completely blocked by the L type Ca 2+ channel antagonists nifedipine, nitrendipine, D600, or diltiazem at subor micromolar concentrations. Measurement of [Ca2+]i in cell bodies using the fluorescent Ca 2+ indicator fura-2 established that optimal growth, similar to that seen in normal medium, was obtained when [Ca2+]i was clamped at resting levels. These levels of [Ca~+]i were set by serum, which elevated [Ca2+]i by ~30 nM, whereas the addition of NGF had no effect on [Ca2+]~. The reduction of [Ca2+]o prevented neurite fasciculation but this had no effect on the rate of neurite elongation or on the number of extending neurites. These results show that neurite outgrowth from NGF-dependent neurons occurs over long periods in the complete absence of Ca 2+ signals, suggesting that Ca 2+ signals are not necessary for operating the basic machinery of neurite