Calcium Entry through L-Type Calcium Channels Is Essential for Neurite Regeneration in Cultured Sympathetic Neurons

Abstract

Previous work showed that a post-neuritotomy rise in [Ca²⁺]_i is required for regeneration. We tested the following hypotheses in cultured sympathetic neurons: (1) blocking L-type channels at the time of injury inhibits regeneration; (2) enhancing Ca²⁺ entry through L-type Ca²⁺ channels enhances regeneration; (3) L-type Ca²⁺ channel distribution is predominantly on the soma and proximal neurites of uninjured and injured neurons. To visualize L-type Ca²⁺ channels and block Ca²⁺ influx, the fluorescent dihydropyridine antagonist, DM-BODIPY, was used. Our results show that regeneration is markedly inhibited by the antagonist when administered 20 min. prior to injury, in the presence or absence of nerve growth factor (NGF) (p < 0.0001). Severe degeneration of proximal and distal neurites was seen 48 h after injury. Regeneration was minimally inhibited by the antagonist when administered 5 min after injury (p < 0.05), but not inhibited when administered 2 or 24 h after injury (p > 0.05). We found that L-type channels are distributed ubiquitously on the soma and neurites of uninjured and injured cells, and on regenerating neurites. The addition of the L-type channel agonist, BayK8644, (1 μM) 20 min prior to injury enhanced neurite length at 24 h post-injury (p = 0.002). Blocking L-type channels did not affect the viability of uninjured or injured cells. For the first time, it has been shown that Ca²⁺ entry through L-type Ca²⁺ channels is essential for post-neuritotomy sympathetic neurite regeneration, and that this effect shows a strict temporal dependency. We also demonstrated that regeneration can be enhanced by increasing Ca²⁺ influx through L-type channels.