Calcium influx is necessary for optimal regrowth of transected neurites of rat sympathetic ganglion neurons in vitro

Abstract

The calcium ion serves many major roles in cellular processes, and therefore, its specific role in the regrowth of neurites after transection has been difficult to define. In the present study the effect of calcium on regrowth of transected rat superior cervical ganglion neurons in culture independent of its effects on neuronal survival was examined. Superior cervical ganglion neurons in culture for 10–14 days were preloaded with the calcium chelator 1,2 bis-(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), and then the neurites were transected. The chelation of calcium inhibited the initiation of regrowth, and also decreased the number of branches in the regrowing neurite. In a second experiment, neurites were transected in zero calcium medium to eliminate calcium influx, and no regrowth was observed for up to 6 h after transection. At this time the medium was changed to 1.8 mM calcium, and the neurites showed regrowth 2 h afterwards. After neurite transection in 1.8 mM calcium there was a rise in calcium in the soma demonstrated with the calcium indicator dye Fluo-3 AM. This rise was attenuated with BAPTA-AM preloading or transection in zero calcium medium. Cultures were also grown in compartmented chambers which allowed isolation of the cell body from the neurite environment, and then the neurites were transected with the neurites in 1.8 mM calcium medium, and the cell bodies immersed in zero calcium medium. Under these conditions the majority of neurons showed a marked delay of initiation of regrowth. From these data we suggest the following about mammalian neurite regrowth after transection. (1) Calcium is necessary for the initiation of regeneration. (2) Calcium entry at the cell body is necessary for optimal initiation of neurite regrowth. (3) Intracellular calcium is necessary for the branching of regrowing neurites.