Ca2+ transients are not required as signals for long-term neurite outgrowth from cultured sympathetic neurons.

Abstract

Abstract.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 sub-or 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 outgrowth.

Nerve Growth Factor is Required for Induction of c‐Fos Immunoreactivity by Serum, Depolarization, Cyclic AMP or Trauma in Cultured Rat Sympathetic Neurons

“…Our results showing that a strong depolarizing stimulus capable of markedly increasing intracellular calcium (Tolkovsky et al, 1990) is not sufficient in itself to induce Fos-IR in isolated sympathetic neurons if NGF has been withdrawn for a few hours, also suggest that Fosexpression cannot be expected to serve as a general marker of neuronal activity in all situations. Although it has been suggested that the differences observed in the propensity of different peripheral neurons to induce Fos-IR in the CNS after stimulation (Hunt et al, 1987) is due to the use of anaesthetics (Dragunow and Faull, 1989), some of the selective effects may indicate a similar dependence on an obligatory enabling signal, such as that provided by NGF.…”

“…Nor is it likely that a reduction of [Ca2+Ii evoked by the removal of NGF(Koike et al, 1989) is the cause of the reduced responsiveness to serum or depolarization because both stimuli elevate [Ca2+]i in the absence of NGF(Koike et al, 1989;Tolkovsky et al, 1990).…”

Calcium transients in growth cones and axons of cultured Helisoma neurons in response to conditioning factors

“…These results confirmed earlier suspicions that calcium transients were not necessary in the initiation of outgrowth. Likewise, recent studies on rat sympathetic neurons demonstrate that calcium transients are not necessary for neurite extension (Tolkovsky et al, 1990). When calcium levels were clamped in these cells, by reducing extracellular calcium with EGTA and depolarizing the cell with high potassium, neurite outgrowth was still initiated in response to NGF.…”

Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons.

“…[Ca2+]i was measured in cell bodies at 370C using a Deltascan system with dual excitation and single emission (Tolkovsky et al, 1990). Excitation at 340 or 380 nm changed every 25 ms, and emission was collected at 511 nm with a photomultiplier tube.…”