EVIDENCE FOR CALCIUM REGULATION OF SPINAL-CORD MOTONEURON DEATH IN THE CHICK-EMBRYO IN-VIVO

We have studied in living chick embryos the effects of an extracellula r calcium load on the induction of apoptosis in spinal cord motoneuron s. The action of a calcium ionophore, A23187, that does not raise extr acellular calcium was also evaluated in order to explore the role of e ndogenous calcium in determining developmentally-regulated cell death of motoneurons. The application of a single dose of 50 mu l of 1.8 M C aCl2 onto the chorioallantoic membrane of E7 chick embryos produces a transient elevation of intraembryonic calcium concentration that was f ollowed by a transitory rise in the number of apoptotic cells in the l ateral motor column. Administration of 250 mu M Of the ionophore A2318 7 (100 mu l), also results in an increase in apoptosis of motoneurons in the lateral motor column on E6 and E7 but this effect is progressiv ely lost following treatment at more advanced stages of development. N either of these effects can be explained by unspecific calcium cytotox icity since they can be inhibited by prior administration of the prote in synthesis inhibitor cycloheximide or the neuromuscular blocking age nt (+)-tubocurarine. After calcium loading, degenerating cells display similar ultrastructural characteristics as during physiologically occ urring motoneuron death and exhibit histochemically detectable DNA fra gmentation. Chronic administration of CaCl2 or A23187 does not reduce the total number of surviving motoneurons at the end of the normal per iod of naturally occurring motoneuron death (E10). It is suggested tha t calcium loading stimulates and accelerates the physiological degener ation of a restricted subpopulation of motoneurons which will undergo the process of natural cell death.